Method and composition for hyperthermally treating cells

ABSTRACT

A method and composition for hyperthermally treating tumor cells in a patient under conditions that affect tumor stem cells and tumor cells.

This application is a Continuation-In-Part of co-pending U.S. Ser. No.14/554,840 filed Nov. 26, 2014, which is a Continuation-in-Part ofco-pending U.S. patent application Ser. No. 14/311,464 filed Jun. 23,2014; which is a Continuation-in-Part of U.S. patent application Ser.No. 13/915,282 filed Jun. 11, 2013; which is Continuation-in-Part ofU.S. patent application Ser. No. 13/665,458 filed Oct. 31, 2012 now U.S.Pat. No. 8,668,935; which is a Continuation-in-Part of U.S. patentapplication Ser. No. 13/610,503 filed Sep. 11, 2012 now U.S. Pat. No.8,709,488; which is a Continuation-in-Part of co-pending U.S. patentapplication Ser. No. 13/527,005 filed Jun. 19, 2012; which is aContinuation-in-Part of co-pending U.S. patent application Ser. No.13/455,237 filed Apr. 25, 2012; which is a Continuation-in-Part ofco-pending U.S. patent application Ser. No. 13/361,786 filed Jan. 30,2012; which is a Continuation-in-Part of co-pending U.S. patentapplication Ser. No. 13/307,916 filed Nov. 30, 2011; which is aContinuation-in-Part of U.S. patent application Ser. No. 13/189,606filed Jul. 25, 2011 now U.S. Pat. No. 8,119,165; which is aContinuation-in-Part of U.S. patent application Ser. No. 13/149,209filed May 31, 2011 now U.S. Pat. No. 8,137,698; which is aContinuation-in-Part of U.S. patent application Ser. No. 12/478,029filed Jun. 4, 2009 now U.S. Pat. No. 7,964,214; which is aContinuation-in-Part of U.S. patent application Ser. No. 11/485,352filed Jul. 13, 2006 now U.S. Pat. No. 7,638,139; which is a Division ofU.S. patent application Ser. No. 10/073,863 filed Feb. 14, 2002 now U.S.Pat. No. 7,101,571; the entirety of each is hereby expresslyincorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to a method and composition forhyperthermally treating cells at a site in the body. More particularly,the present invention relates to a method for treating cells at a targetsite in the body, such as at a lens capsule of an eye, tumors, andexudative or wet age related macular degeneration (AMD) by applyingthermal energy to the target site to heat the cells to a temperaturewhich will kill the cells or impede cell multiplication withoutexceeding the protein denaturation temperature of the tissue.

BACKGROUND OF THE INVENTION

Several techniques currently exist for treating cells at a selected sitein the body with heat or chemicals to kill or impede multiplication ofthose cells to prevent undesired cell proliferation. For example,numerous types of chemotherapy drugs exists which, when injected into atumor or delivered systemically to a patient, attack and kill cancerouscells to prevent them from further multiplying. However, unless thetreatment affects the tumor stem cells that have mutated to result inuncontrolled tumor growth and metastasis, treatment will not beeffective. Stem cells are pluripotential undifferentiated cells. Tumorstem cells are frequently located in the bulk tumor mass, are involvedin tumor metastasis, and often elude detection. Over time, stem cellsbecome resistant to standard chemotherapy regimens by constant geneticmutations that confer resistance.

Thermal radiation techniques can also be used to kill cancerous or otherundesired cells. Cell death begins to occur when the cells are heated toa temperature of about 5° C. or more above the normal body temperatureof 37° C. Applying thermal radiation to a localized site in the body,such as a tumor or other area containing undesired cells, can heat thecells at the site to temperatures in excess of 60° C. Such hightemperatures causes a phenomenon known as protein denaturation to occurin the cells, which results in immediate cell death. Accordingly,thermal radiation therapy has been suitable in successfully treatingcertain types of cancers and other diseases involving uncontrolled cellgrowth.

Other types of heating techniques, such as the use of probes orcatheters to provide localized heat to a site of interest also exist.Like thermal radiation therapy, these techniques also heat the cells toa temperature sufficient to cause protein denaturation in the cells tothus kill the cells quickly.

Photosensitive chemicals are also used to kill cells at certain sites ofinterest in the body. For example, a photosensitive chemical can beinjected directly into a site of interest to expose cells at that siteto the chemical. A light emitting source, which emits light at awavelength that will activate the photosensitive chemical, is thenfocused on the site of interest. Accordingly, the light activates thephotosensitive chemical that has been absorbed by or is otherwisepresent in the cells of interest. The activated chemical kills thecells, which thus prevents undesired cell proliferation.

Although the techniques mentioned above can be suitable for preventingcertain types of cell proliferation at certain sites in the body,several drawbacks with these techniques exist. For example, often theuse of chemotherapy drugs alone to treat a tumor or cancerous site isinsufficient to kill the undesired cells. The only current treatmentsdirected at tumor stem cells are heavy doses of ionizing radiation,thermal radiation or thermotherapy, or chemotherapy. Moreover, thechemotherapy drugs and other treatments also indiscriminately kill manynormal healthy cells along with the cancerous cells, which can adverselyaffect the patient's health and are frequently ineffective against latestage cancers.

The use of ionizing radiation in conjunction with chemotherapy can havea more detrimental effect on the cancerous cells. However, as withchemotherapy, ionizing radiation often kills normal healthy cells, suchas those in front of or behind the site of interest, along with thecancerous cells. Moreover, the intense heating of the cells can causethe cells to coagulate and thus block the capillaries at the site ofinterest. The blocked capillaries therefore prevent chemotherapy drugsfrom reaching the site of interest.

One example of a method of chemically treating a target site isdisclosed in U.S. Pat. No. 6,248,727 to Zeimer. This method delivers aliposome containing a fluorescent dye and tissue-reactive agent. Theliposome is administered intravenously to flow to the locus in the eyeof the patient and the site is non-invasively heated to release the dyeand the tissue-reactive agent. The dye is fluoresced to observe thepattern of the fluorescence. The tissue-reactive agent is activated tochemically damage and occlude the blood vessel. The liposomes areselected to release the dye at a temperature of 41° C. or less withoutcausing thermal damage to the blood vessel.

In addition, the above techniques have not been used to prevent unwantedcell proliferation at certain locations in the eye, such as at theretina or at the lens capsule. Because the retina is very sensitive,conventional ionizing radiation techniques can be too severe to treatcancerous cells on, in or under the retina.

Also, after cataract surgery, a phenomenon known as capsularopacification and, in particular, posterior capsular opacification canoccur in which the epithelial cells on the lens capsule of the eyeexperience proliferated growth. This growth can result in the cellscovering all or a substantial portion of the front and rear surfaces ofthe lens capsule, which can cause the lens capsule to become cloudy andthus adversely affect the patient's vision. These cells can be removedby known techniques, such as by scraping away the epithelial cells.However, it is often difficult to remove all of the unwanted cells.Hence, after time, the unwanted cells typically will grow back, thusrequiring further surgery.

Accordingly, a need exists for a method for hyperthermally treatingtissue and preventing unwanted cell proliferation at sites in the body,especially at sites in the eye such as the retina, choroid and lenscapsule, which does not suffer from the drawbacks associated with theknown techniques discussed above. The method should also treat tumorstem cells to target and eradicate a tumor source and eliminate or slowtumor metastasis. The method should also target and damage the specifictumor-associated vasculature, in effect, starving the tumor of itsnutrient supply.

SUMMARY OF THE INVENTION

The present invention is directed to a method of hyperthermally treatingtissue by heating the tissue above a temperature which kills cells inthe tissue. In particular, the invention is directed to a method ofheating tissue above a temperature effective to treat the tissue withoutdenaturing the protein. The present invention also relates to a methodand composition for hyperthermally treating cells in the eye withsimultaneous imaging.

One embodiment of the method targets tumor stem cells by combiningchemotherapy with thermotherapy to target tumor stem cells while leavingnormal cells either unaffected or minimally effected by the therapy.This embodiment of the method uses increases in temperature to firstprime the tumor stem cells, and then to kill the tumor stem cellssynergistically with thermotherapy. In one embodiment, nanoparticlesthat are coated with or otherwise contain antibodies that specificallytarget certain cells and/or cell types, e.g., tumor cells. This helps tominimize or eliminate chemotherapy adverse effects on non-tumor bearingvital organs such as liver, kidney, gut, heart, central nervous system,while providing supra-therapeutic doses of the chemotherapy drugs totumor cells, both local and metastatic, and stem cells. In this way, theduration of chemotherapy administered to a patient may be reducedwithout compromising effective treatment.

One embodiment of the method targets tumor stem cells by combininglocalized internal ionizing radiation therapy with thermotherapy totarget tumor stem cells and to destroy their associated vasculature,while leaving normal cells and non-tumor vasculature either unaffectedor minimally affected by the therapy. This embodiment of the method usesincreases in temperature to first prime the tumor stem cells, and thento kill the tumor stem cells synergistically with thermotherapy, anduses localized internal ionizing radiation therapy to destroyvasculature associated with the tumor. In one embodiment, nanoparticlesthat are coated with or otherwise contain antibodies that specificallytarget certain cells and/or cell types, e.g., tumor cells, also containa radioactive isotope. The increased temperature from the thermotherapykills the tumor stem cells, both local and metastatic. The localizedinternally administered radiotherapy damages the proximate and/oradjacent endothelial cells of the vasculature feeding the tumor cells,both local and metastatic. The embodiment of the method may be used in apatient receiving anti-vascular endothelial growth-factor therapy toprovide an additional source of vessel damage. This combination therapyincreases the likelihood of eradication of tumor cells and stem cellsassociated with the tumor by additionally damaging or obliterating thevessels that provide their nutrient supply. The result is more robusttherapy and increased chances for patient survival.

Accordingly, a primary aspect of the invention is to provide a methodfor heating tissue at least to a temperature sufficient tohyperthermally treat the tissue.

Another aspect of the invention is to provide a method of hyperthermallytreating tissue to a temperature sufficient to kill cells in the tissueand at a temperature below the protein denaturization temperature of thetissue.

A further aspect of the invention is to provide a method ofhyperthermally treating tissue, where the tissue includes or is providedwith a temperature indicator to indicate a hyperthermally effectivetemperature of the tissue.

Still another aspect of the invention is to provide a method ofhyperthermally treating tissue where a temperature indicator compositionis introduced into the tissue or bloodstream near the tissue to indicatea tissue temperature effective to hyperthermally treat the tissue and atemperature indicator to indicate a tissue temperature above a proteindenaturization temperature of said tissue.

A further aspect of the invention is to provide a method ofhyperthermally treating tissue by introducing a temperature indicatorinto the tissue and heating the tissue to a temperature where thetemperature indicator can be detected. In a preferred embodiment, thetemperature at which the indicator can be detected is a temperatureeffective to hyperthermally treat the tissue and is at a temperaturebelow the protein denaturization temperature.

A further aspect of the invention is to provide a method of heating anddetecting a temperature of a tissue between a first temperature and asecond temperature. The method introduces a temperature indicator intothe tissue. The temperature indicator includes a first dye that can bedetected at the first temperature to indicate that the first temperaturehas been reached, and a second dye that can be detected at the secondtemperature to indicate that the second temperature has been reached.

Still another aspect of the invention is to provide a temperatureindicating composition for introducing into a tissue to be thermallytreated. The composition includes a first dye encapsulated in a heatsensitive liposome where the first dye is releasable at a temperatureeffective to hyperthermally treat the tissue and at a temperature belowthe protein denaturization temperature. The composition also includes asecond dye encapsulated in a second liposome where the second dye isreleasable at a temperature at or above the protein denaturizationtemperature.

Another aspect of the invention is to provide a method to hyperthermallytreat tissue to kill the tissue cells substantially without proteindenaturization of the tissue where the tissue includes a heat sensitiveliposome containing a temperature indicating dye and a temperatureactivated bioactive compound. The tissue is heated to release the dyefrom the liposome to indicate a thermally effective temperature to killcells in the tissue at a temperature below the protein denaturizationtemperature. The heat applied to the tissue simultaneously releases thebioactive compound to treat the tissue.

Another aspect of the invention is to provide labeled nanoparticles,either alone or conjugated with activatable cell-penetrating peptides(ACPPs) to assist in localizing and destroying cancer cells or tumortissue remaining after surgical excision of a tumor. Another aspect ofthe invention relates to ACPP-conjugated nanoparticles that can also beadapted to attach to normal cell specific receptors in order to stainand treat the tissues. These cells can be as involved with vasculaturesuch as myofibroblasts becoming activated after stent surgery; or nervecells that create neuromas. Another aspect of the invention relates tothe treatment or debulking of other unwanted masses, such as fibromas,meningiomas, adenomas, or degenerative cells causing Parkinson's Diseaseor of nerves involved in transmitting pain, e.g., for patients inchronic pain. Another aspect of the invention is to treat malignanttumors, such as neuroblastoma, melanoma, skin, breast, brain kidney,lung, intestinal, and genitourinary, bone, gland, and blood cancers.Another aspect of this invention is to target normal cells of the bodyresponsible for an immune response, such as lymphocytes, etc., thatprevent an organ transplant. Another aspect of the invention is to treatundesirable vascular rupture and bleeding.

Another aspect of the invention is to target and damage tumor cells,tumor stem cells, and endothelial cells of tumor-associated vessels,e.g., blood vessels, lymphatics.

Another embodiment of the invention is to provide immunotherapy incombination with thermal therapy to a target site. Thermotherapy isprovided by administering a composition comprising a targeting agentand/or antitumor-antibody-labeled nanoparticle where the nanoparticleforms a targeting agent and/or antibody labeled nanoparticle-cellcomplex at a tumor site, exposing the patient to an energy source wherethe nanoparticles respond by expanding thus generating an acousticsignal, measuring the acoustic signal and relating the measured signalto a temperature of the nanoparticle-cell complex, and controlling thetemperature of the nanoparticle-cell complex from about 39° C. to about58° C. based on the acoustic signal to hyperthermally damage or killcells in the tumor; and providing immunotherapy by providing animmunotherapy agent associated with the nanoparticles, providing animmunotherapy agent not associated with the nanoparticles, and/orproviding a therapeutic concentration of autologous leukocytes to thepatient, where immunotherapy enhances a patient's immune response. Inspecific embodiments, thermotherapy is performed in the absence of acontrast agent, substantially simultaneously with, prior to, orsubsequent to immune therapy. The immunotherapy may reduceimmunosuppression. The thermotherapy may supplement chemotherapy and/orradiation therapy. A marker may be evaluated to assess a therapy effect.The nanoparticle may be associated with a member of specific bindingpair such as streptavidin-biotin pair, a cellular receptor-agonist orantagonist pair, an enzyme-substrate pair, and/or an antibody-antigenpair. The nanoparticle may be associated with a virus, e.g., a modifiedvirus, a tumoricidal virus, and/or an adeno-associated virus (AAV).

The various aspects of the invention are basically attained by providinga method of hyperthermally treating tissue in an animal. The methodcomprises the step of introducing a temperature indicating substanceinto the bloodstream of the animal to flow through a target site. Thetemperature indicating substance includes a fluorescent dye encapsulatedwithin a heat sensitive liposome. The fluorescent dye is releasable fromthe liposome at a temperature of at least 41° C. A heat source isapplied to the target site and the target is hyperthermally heated to atleast 41° C. to release and fluoresce the dye and to hyperthermallytreat the target site for a time sufficient to kill cells in the tissue.

The aspects of the invention are also attained by providing a method ofdetecting a threshold temperature and of hyperthermally treating tissuein an animal. The method comprises the step of introducing a firstfluorescent dye encapsulated in a first heat sensitive liposome into thebloodstream of an animal in a location to flow through a target site inthe animal. The first fluorescent dye is releasable from the first heatsensitive liposome at a temperature of at least 41° C. The target siteis heated to a temperature to release the first fluorescent dye and thefirst fluorescent dye is fluoresced to indicate and visualize a tissuetemperature of at least 41° C. Heating of the target site is continuedat a temperature of at least 41° C. for a time sufficient tohyperthermally treat the tissue.

Another aspect of the invention is a method of targeting tumor stemcells for therapy in a patient needing the therapy, e.g., a cancerpatient either simultaneously undergoing chemotherapy or havingpreviously undergone chemotherapy. The method administers chemotherapywith thermotherapy, with thermotherapy being a stepwise increase intemperature that, at relatively lower temperatures, primes the tumorcells, including tumor stem cells, for increased susceptibility to thechemotherapy. Then at relatively higher temperatures, the method killsthe primed tumor stem cells with a synergistic combination of thechemotherapy and the thermotherapy.

Another aspect of the invention is a method of targeting tumor cells,tumor stem cells, and endothelial cells of tumor-associated vessels toselectively destroy both the tumor as well as the vessels feeding thetumor by combining thermal therapy with localized internallyadministered ionizing radiation therapy.

The aspects of the invention are further attained by providing a methodof hyperthermally treating tissue of an animal. The method comprises thestep of introducing a temperature indicating substance into thebloodstream of the animal to flow through a target site. The temperatureindicating substance includes a first fluorescent dye encapsulated in afirst temperature sensitive liposome. The first fluorescent dye isreleasable from the first liposome by heating to a temperature of atleast 42° C. A second fluorescent dye encapsulated in a secondtemperature sensitive liposome is also included. The second fluorescentdye is releasable from the second liposome by heating to a temperatureof at least 50° C. The target site is heated to a temperature of atleast 42° C. The first fluorescent dye is fluoresced to indicate aneffective temperature for hyperthermally treating the tissue withoutreleasing the second fluorescent dye from the second liposomes.

Another aspect discloses a method of delivering particulate material,e.g., nanoparticles, into mammalian cells and/or tumor cells throughpinocytic uptake and/or using various channels. In one embodiment, theparticulate material is delivered to the nuclear membrane, whichtypically has a pore size<100 nm in diameter. In one embodiment, thepores and/or pinocytic vesicles etc. permit the nanoparticles, smallerthan 100 nm, to be taken up into the cytoplasm or the nucleus of thecells/bacteria. The nanoparticles are coated with or contain an antibodyand/or drug. The non-magnetic and/or magnetic nanoparticles, once takenup by the cell/bacteria, are heated selectively by an external sourcesuch as electromagnetic energy or a reversible magnetic field, andimaged using either a photoacoustic technology or MRI.

One embodiment is a method of providing therapy by administering, to apatient in need of therapy, nanoparticles coated or otherwise associatedwith an antibody to specific cells, under conditions sufficient topermit antibody accumulation at a tissue target site, radiating thetarget site with an energy source to penetrate into the tissue targetsite to controllably heat the nanoparticles and generate thermal energyto induce a photoacoustic signal or sound wave from the nanoparticles,using a processor to control the amount of thermal energy delivered atthe desired temperature to the target site, recording the temperatureand photoacoustic signal or sound wave from the target site or from oneor more multiple locations, and amplifying and processing the recordedphotoacoustic signal or sound waves to generate a computationaltomographic image of the nanoparticles at the tissue target site.

In this embodiment, imaging generates high-resolution two- orthree-dimensional photoacoustic images. In one embodiment, the antibodyis an anti-tumor antibody, the nanoparticles are attached to tumorcells, and the method generates a two- or three-dimensionalphotoacoustic image of the tumor. In one embodiment, the antibody isdirected to a receptor on a normal cell of an organ, the nanoparticlesare attached to the normal cells, and the method generates a two- orthree-dimensional photoacoustic image of an organ regardless of locationof the organ in the body. Imaging may use radiofrequency, microwave,ultrasound, and/or focused ultrasound. Imaging may be by photoacoustictemperature imaging combined with ultrasound, focused ultrasound,magnetic resonance imaging (MRI), functional MRI (fMRI), computedtomography (CT), positron emission tomography (PET), OCT, andalternating magnetic field imaging, resulting in enhanced imageacquisition, resolution, visibility, distinction, and/or utility.

In this embodiment, the processor communicates the temperature from thephotoacoustic source to the energy source to control the amount andduration of energy delivered to a desired temperature. The photoacousticsignal or sound wave is recorded and produced as each of a thermal graphof the target site, and as an image in one-dimension, two-dimensions, orthree-dimensions.

In this embodiment, the energy source to radiate the target site may beelectromagnetic radiation, ultraviolet radiation, visible light,infrared light, radiofrequency waves, microwaves, focused ultrasound,and/or alternating magnetic field radiation. A combination of energysources decreases the amount of energy required for each unit or source,e.g., lasers of various wavelengths, radiofrequency waves, microwaveswith focused ultrasound, and microwaves with alternating magnets. Energyis applied from multiple sites or using multiple energy sources thusminimizing or preventing pain to the patient and overheating of thetarget site. The applied energy may be continuous, intermittent,oscillatory, and/or pulsed. Energy applied in an oscillatory or pulsedmanner reduces thermal damage to normal cells while sufficiently heatingcells to which the nanoparticles are attached to the desiredtemperature. Energy may be applied intermittently as an alternatingmagnetic force to heat to the nanoparticle-cell complex, and imagingmeasures the temperature of the heated tissue and images the targetsite. In this embodiment, supramagnetic ferric oxide and/orsupraparamagentic ferric oxide nanoparticles, nanotubes, and nanowiresare particularly preferred.

In this embodiment, the target site may be maintained at temperaturesranging from 39° C. to 48° C., 37° C. to <60° C., 37° C. to 41° C., 42°C. to 46° C., 47° C. to 50° C., and 50° C. to 58° C.

In one embodiment, nanoparticles are attached to tumor cells, the tumorcells are selectively or preferentially treated over normal non-tumorcells, and the thermal energy is applied from multiple locationscovering an area of 1° to 90°, up to 180°, or greater than 180° of thecircumference of the target tissue.

The patient undergoing therapy by this embodiment of the method may havea benign or malignant lesion, and/or an infection. Cells may bepreloaded stem cells, macrophages, dendritic cells, and/or exosomes ofdendritic cells, with the method locating or tracking the cellsthroughout the body, and imaging the cells accumulation in vessels ortissues at a site of inflammation.

In this embodiment the nanoparticles may be synthetic, organic,non-organic, non-magnetic, magnetic, paramagnetic, diamagnetic,supramagnetic, non-magnetic, mesoporous carbide-derived carbon, ironoxide nanoparticles with gold, graphene oxide and mesoporous siliconenanostructures, carbon, quantum dots, nanoshells, nanorods, nanotubes,and/or nanowires. The nanoparticles may be liposomal nanoparticles,liposome-PEG nanoparticles, micellar polymeric platform nanoparticles,L-adenine nanoparticles, L-lysine nanoparticles, PEG-deaminasenanoparticles, polycyclodextrin nanoparticles, polyglutamatenanoparticles, calcium phosphate nanoparticles, antibody-enzymeconjugated nanoparticles, polymeric lipid hybrid nanoparticles,nanoparticles containing a combination of two-three elements such asgold, gold-iron oxide, iron-zinc oxide, metallic nanoparticles,polylacticglycolic acid nanoparticles, ceramic nanoparticles, silicananoparticles, silica crosslinked block polymer micelles, albumin-basednanoparticles, albumin-PEG nanoparticles, and/or dendrimers attachedmagnetic or non-magnetic nanoparticles. In one specific embodiment,nanoparticles are iron oxide coated with an oligosaccharide.Nanoparticles may be coated with thermosensitive polymers carryingagents, e.g., anti-infectives, chemotherapeutics, anti-VEGFs,anti-EGFRs, hormones, immunosuppressants, immunostimulatory agents, DNA,RNA, siRNA, genes, and/or nucleotides. The nanoparticles may be coatedor otherwise associated with biocompatible molecules, e.g., PEG, biotin,CPP, ACPP, dendrimers, dendrimers conjugated with poly beta amine,and/or small organic molecules.

In this embodiment, the antibody may target a circulating cell or anon-circulating cell fix in an organ or specific location, and the cellmay be either benign or malignant. The antibody may be, e.g., abciximab,adalimuab, alemtuzumab, brentuximab, blimubab, canakinumab, alemtuzumab,cituximab, cetrolizumab, daclizumab, denosumab, efalizumab, gemtizumab,glimumab, lipilimumab, infliximab, rituximab, muromonab, oftamumab,palivizumab, panitumab, ranibizumab, tositumomab, and/or rastuzumab.

In a specific embodiment of this general embodiment, the method isprovided to the patient with a method for enhanced cell penetration ofthe antibody-nanoparticle complex, e.g., application of ultrasoundand/or electroporation. In another specific embodiment, the method usesa combination of CPP and poly beta amine to enhance cell penetration ofthe antibody-nanoparticle complex. This embodiment may reduce the totalamount of energy applied to achieve therapy of cells at the target sitewhile sparing normal cells.

In another specific embodiment of this general embodiment, the patientis undergoing cancer immunotherapy and the method provides an agent tothe patient to result in enhanced immunogenicity, e.g., an inhibitor toCHK1, CHK2, CHM1, and/or CHM2.

In another specific embodiment of this general embodiment, patient cellsare grown in culture with the nanoparticles, and the cultured cells withnanoparticles incorporated are injected into the patient. The cellsgrown in culture may be stem cells, macrophages, dendritic cells,lymphocytes and other leukocytes, and/or exosomes from dendritic cells.The nanoparticles can be imaged and traced in the patient, e.g., one orseveral organs, and can result in indirectly imaging an inflammatoryprocess in tissues of the organ.

The nanoparticles size may range from 1 nm-800 nm, 20 nm to 200 nm, 1nm-20 nm, or 1 nm-10 nm.

In another specific embodiment of this general embodiment, the targetsite contains a lesion or pathology, and the nanoparticles contain apolymeric coating that itself contains a medicament that is releasedwhen the temperature is 40° C.-47° C. resulting in combined thermal andmedicament therapy to the lesion.

The another specific embodiment of this general embodiment, the patientundergoing therapy is treated simultaneously or substantiallysimultaneously with hemofiltration, hemoadsorption, mesoporouscarbide-derived carbon, or another type of agent to prevent a cytokinestorm.

In another specific embodiment of this general embodiment, photoacousticimaging in combination with MRI, ultrasound, or light results inenhanced image distinction of a structure without heating the structuresignificantly beyond 37° C.-39° C.

In another specific embodiment of this general embodiment, the methodcreates a photoacoustic image of an internal structure withradiofrequency wave energy or microwave energy alone. An antibody-coatednanoparticle is targeted to a tumor or structure that, becausenanoparticles of 1 nm are significantly smaller than a micron, twonanoparticles are separated only by <2 nm-3 nm, using thermal energy,creating distinct separate signals that are electronically enhanced and,using photoacoustic technology, two points separated by that distanceare calculated and measured. This provides enhanced imaging beyondoptical resolution of a structure using imaging by, e.g., light, MRI,CT, and/or PET.

These and other aspects of the invention will become apparent to oneskilled in the art in view of the following detailed description of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of one embodiment of the invention showinga probe for hyperthermally treating tissue and visualizing a dye in thetarget site.

FIG. 2 illustrates therapy of a patient with a choroidal tumor.

FIG. 3 illustrates electromagnet administration non-invasively, throughthe conjunctiva to the tumor.

FIGS. 4A and 4B illustrate one embodiment after placement of the magnetfor a tumor affecting the lower leg and with a horseshoe-shapedelectromagnet configuration.

FIG. 5 illustrates one embodiment after placement of the magnet for afacial skin tumor.

FIG. 6 shows the relationship between the photoacoustic system, theprocessor, and the energy delivery system.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method and composition forhyperthermally treating tissue. In particular, the invention is directedto as method for heating tissue above a temperature effective to killtissue cells or inhibit multiplication of cells below the proteindenaturization temperature of the tissue.

The method of the invention introduces a composition into thebloodstream or other system of the body in a location to flow into orthrough a target site to be treated. In one embodiment, the compositionis introduced into the lymphatic system. A heat source such as generatedby radiation energy is applied to the target site to heat the tissue inthe target site for a time sufficient to hyperthermally treat the tissueand activate the composition. As used herein, the term “hyperthermal”refers to a temperature of the cell or tissue that kills or damages thecells without protein denaturization.

The composition may contain a temperature indicator that is able toprovide a visual indication when a minimum or threshold temperature isattained that is sufficient to hyperthermally treat the tissue. It is afeature of the invention to provide a method of heating tissue in atarget site to a hyperthermally effective temperature and to provide avisual indication that a temperature of at least 41° C., and preferablyat least 42° C. is attained. In one embodiment, the composition includesa second temperature indicator to provide a visual indication when aprotein denaturization temperature is attained thereby providing anindication that a maximum desired temperature is exceeded. The heatsource can be applied to the tissue so that the composition provides anindication that a thermally effective temperature is attained that isbelow the protein denaturization temperature of the tissue.

In one embodiment of the invention, the method introduces a compositionto a target site, where the composition includes a fluorescent dye thatis encapsulated in a heat sensitive particle, such as a liposome. Thedye is a fluorescent dye that can be excited to fluoresce and beobserved or visualized by the operator. Preferably, the heat sensitiveliposomes are formed to rupture or release the fluorescent dye at atemperature at least equal to the temperature necessary to kill cells inthe tissue and at a temperature below the protein denaturizationtemperature. The composition containing the heat sensitive liposomesencapsulating the fluorescent dye is introduced into the bloodstream toflow to or through the target site. The amount of the liposomecomposition is introduced in an amount effective to be released in ornear the target site and to excited and visualized by the exciting lightsource and the visualizing device. The composition containing the dyecan be injected in a single dose into the bloodstream or injectedcontinuously to supply a continuous flow of the composition through thetarget site. The amount of the composition introduced can vary dependingon the target site and the length of time that the dye is to be excited.A light or energy source is continuously applied to the target site toexcite the dye and to cause the dye to fluoresce when released from theliposomes. An imaging device is used to capture the fluorescing lightfrom the dye to provide a visual indication that the dye is released.The release temperature of the liposomes are selected to release the dyeat a predetermined temperature so that when the dye is fluoresced andvisualized, the visualization provides the operator with an indicationthat the release temperature in the target site has been attained. Inone embodiment, the liposome composition is injected into the bloodstream so that the composition is able to provide a continuous supply ofthe dye for fluorescing during the hyperthermal treatment. In thismanner the operator is provided with a continuous indication that asufficient temperature is being maintained.

The method of the invention is primarily directed to a method of heatingtissue and cells in the tissue of an animal, particularly a humanpatient, at least to the temperature sufficient to kill or damage thecells. Cell death or cell damage is known to occur when the tissue cellsare heated to a temperature of about 5° C. above the normal bodytemperature of 37° C. Therefore, the method of the invention heats thecells in the tissue to a temperature of about 41° C., and preferably atleast 42° C. for a time sufficient to kill or damage the cells.Preferably, the heat source is applied to minimize unnecessary damage tothe surrounding cells and tissue.

In one embodiment of the invention, the tissue is heated to atemperature of at least 41° C. and preferably in the range of at leastabout 42° C. to about 50° C. Heating the tissue to at least 42° C.ensures that a sufficient temperature is obtained to thermally treat thetissue and the cells effectively. Preferably, the tissue is heated to atemperature below the protein denaturization temperature of the tissue.Protein denaturization begins to occur at about 50° C. to 51° C. andoccurs rapidly at temperatures of about 60° C. Preferably, the tissue isheated to a temperature of less than 60° C. and more preferably to atemperature of about 50° C. or less.

In one preferred embodiment, the tissue and the cells are heated to atemperature of about 47° C. to about 49° C. for a time sufficient tokill or damage the cells without protein denaturization. The length oftime that the tissue is heated will depend on the location of the targetsite, the size and dimensions of the target site, the desired depth ofpenetration of the heat and the desired extent of thermal treatment- ordamage of the tissue and cells in the target site. Typically, the heatsource is applied for several minutes. In one embodiment, the heatsource is applied for about 1 to 15 minutes, and typically about 5 to 10minutes.

The heat source can be applied to a variety of the areas in the bodywhere the hyperthermal treatment is desired. The target site can betumors, organs, muscles and soft tissue. Examples of a target siteinclude blood vessels and arteries, esophagus and eyes. In oneembodiment, the method is suitable for hyperthermally treating theepithelial cells on the lens of the eye after cataract surgery. Othertarget sites include the retina and the choroid.

In other embodiments the target site may be cell components of variousorgans. The organs may be healthy or may contain tumors, eithermalignant or benign. The following are representative, not limiting,examples of cell components on which the inventive method may beapplied: tumors of the central nervous system (CNS), various layers ofskin and its underlying support structures, intestinal tract, kidney,urinary tract, female and male reproductive system organs, boneincluding bone marrow, circulatory system components including theheart, blood vessels, and circulating malignant cells, the lymphaticsystem including lymph nodes and vessels, and the respiratory system.

In one embodiment, the compositions including, e.g., gold orferromagnetic nanoparticles, as described below, are injected throughthe nipple. The injected composition travels through the duct leadingdown from the nipple ending in glands, i.e., acini aggregated intolobules. This mode of injection may also introduce the composition intothe lymphatic system, particularly if injected into the breast stroma.This embodiment may be used to treat breast cancer, as well as cancersin the associated lymphatic tissue. In addition, this embodiment may beused as a prophylactic treatment to obliterate or substantially reducethe breast glands' secretary epithelium in patients who exhibit geneticpredisposition to breast cancer.

In various embodiments, the described compositions are introducedthrough any accessible cavity, such as oral, respiratory, orgenitourinary cavities. The compositions may be introduced by needleinjection or via a catheter. In one embodiment, the described methodsmay be used to treat cervical or bladder cancer, including early stagesof the diseases.

In one embodiment, cell proliferation in tumors that would otherwise beexcised from a body surface or removed from an internal body site aretreated by the inventive method. Treatments involves both ameliorationand/or tumor debulking, i.e., reducing the tumor mass. In oneembodiment, the inventive method is used to augment surgical removal ofa tumor. Tumors that are relatively large are often surgically removed,even if the tumor is determined to be non-malignant, because of thespace-occupying nature of the tumor and/or the stress on other organs.For examples, stress on adjacent organs, such as liver and kidneys, canresult in the potential for hepatic and nephrotic complications.

During surgical tumor excision or removal, it is important to ensure thetumor is completely removed, yet the tumor margins are difficult torecognize and determine. Typically, complete tumor removal requiresmultiple biopsies, during the surgical procedure itself and while thepatient is under anesthesia, from the edges of the tumors. The biopsiesare histologically or otherwise evaluated in real time by a pathologist.This very time consuming task prolongs surgery, may put the patient atincreased risk, and definitive pathology results may take hours toobtain. Often, even after careful excision of the tumor, subsequenthistologic studies of the entire lesion show that not all margins oredges are free of tumor tissues, resulting in patient anxiety, increasedcost for subsequent surgery, and heightened risk of post-surgicalcomplications. Such uncertainty of complete tumor and/or cancer cellremoval makes subsequent medical treatment difficult to determine, e.g.,whether to remove lymph nodes located in the area of the tumor, etc.

Malignant tumors have a preferred location in the body from which theygrow from the initial mother cell. They then metastasize in otherorgans. The initial tumors are seen in any organ of the body (e.g., eye,brain, skin from which retinoblastomas, gliomas, melanomas occur; alsoprostate, colon and other intestinal tumors, pancreas, thyroid, etc.Tumors can grow from any type of tissue (e.g., ectoderm, mesoderm,endoderm, neuro-ectoderm). Using the eye as one example, melanomas canoriginate from the uveal tract, conjunctiva, orbit, etc. Melanomas ofthe choroid, ciliary body, and iris grow very slowly, in contrast tocutaneous melanomas, and remain in place before metastasizing. Morerapidly growing melanomas preferentially metastasize in the liver, butare not limited and may metastasize to any organ. Prostate cancerpreferentially metastasizes to bone. Colon cancer preferentiallymetastasizes to liver. Some tumors, such as basal cell carcinoma,gliomas, meningioma, etc. grow locally but damage vital organs.

It is thus desirable to treat the early diagnosed tumors locally andwith a more intense therapy such as using ferromagnetic nanoparticles,with or without a single or combination of drugs, that can be attractedto a locally positioned magnet, either external or internal. Placementof a naturally occurring magnet or electromagnet, with a stable magneticfield, adjacent to or over the tumor area attracts and concentrates thecirculating ferromagnetic particles to that specific location duringtheir circulation in the blood after administration (e.g., injection)into the circulatory system, body cavity, etc., or at the site ofadministration, e.g., when topically applied to the skin, mucosa, etc.

In this embodiment, using a magnet to localize ferromagnetic particlesat a tumor or lesion site, or even using whole body therapy,thermotherapy is controllably applied to a patient to treat a localizedtumor and metastasized lesions.

Activatable cell-penetrating peptides (ACPPs) that are labeled withfluorescent polycationic cell-penetrating peptide (CPP) coupled by acleavable linker to a neutralizing peptide have been developed andutilized to visualize tumors during surgery. Similarly there are othertumor specific biomarkers that are known to one skilled in the art, usedfor breast tumor, genitourinary tumors, lung tumors, head and necktumors, gastrointestinal tumors, brain tumors, etc., as only onenonlimiting example, disclosed athttp://www.medscape.org/viewarticle/725989. ACPP conjugated todendrimers (ACPPDs) and gadolinium chelates can allow MRI visualizationof whole body tumors, permitting thermotherapy if the magnetic orgadolinium nanoparticles are labeled with ACPPD. However, grossobservation of a labeled tumor does not guarantee that all the tumormargins will be visible or will removed. One reason is that tumormargins may contain sparse numbers of dispersed cancerous cells. Anotherreason is that cancerous cells may be embedded more deeply in the tissueand thus not amenable to labeling (e.g., staining) and/or visualization.Thus, while such a method helps the surgeon during a surgical procedure,it does not eliminate the need for a biopsy from the tumor margin oredge.

The inventive method provides safe surgical debulking of a large tumor,while concomitantly providing that the tumor margins or edges are freeof viable cancerous cells, or that such cancerous cells are non-viable.The method removes and treats non-malignant tumors, damaged cells suchas nerve cells, or normal cells involved in or affected by adverseimmune reactions in organ transplant procedures.

One embodiment is a composition of a nanoparticle that is conjugated toan ACPP. The nanoparticle may be cleavably conjugated to the ACPP by,e.g., a linker (e.g., at least ethylene glycol moiety or a(poly)ethylene glycol moiety). In one embodiment, the nanoparticle islabeled with a label such as a fluorescent moiety, chemiluminescentmoiety, etc. In one embodiment, the ACPP is labeled with a polycationiccell-penetrating peptide (CPP).

In one embodiment of the method, the above-described composition isprepared with any biocompatible excipient and is administered to apatient. In one embodiment, a regimen of low dose medication isprescribed to enhance the therapeutic effect of the inventive methodincluding but not limited to at least one antiproliferative agent, e.g.,cisplatin, carboplatin, tetraplatin, iproplatin, adriamycin, mitomycinC, actinomycin, ansamitocin and its derivatives, bleomycin, Ara-C,daunomycin, metabolic antagonists such as 5-FU, methotrexate, isobutyl5-fluoro-6-E-furfurylideneamino-xy-1,2,3,4,5,6hexahydro-2,4-dioxopyrimidine-5-carboxylate, melpharan, mitoxantrone,lymphokines, etc.

One embodiment provides a method of drug delivery and thermal damage toa target in a patient in need thereof. This embodiment provides fourranges of thermotherapy at the target site. The target may be aparticular site in the body, e.g., a tumor site. The target may be anorganism, e.g., a bacterial target. Each temperature range results in aspecific effect, as subsequently described. The overall result of thisembodiment is specific drug delivery and specific thermal treatment ofthe target.

This embodiment is based on the knowledge that pores in bacterialmembrane increase specifically during bacterial cell growth. Pores canserve as a conduit for transfer of genes and plasmid DNA from otherbacteria that can effect cell survival, e.g., render the bacteriadrug-resistant. Bacteria can be engineered to produce proteins, e.g.,insulin, by transmitting the genes encoding such proteins to thebacteria interior. One skilled in the art appreciates that the pore sizeand number of pores increase with a cell temperature increase in therange of about 2° C. to about 28° C. At a temperature of greater thanabout 40° C., bacteria become very vulnerable, e.g., they becomeparticularly sensitive to drugs and frequently die. While the samemechanisms take place in mammalian cells, mammalian uptake occurs moreby pinocytosis.

One embodiment is a method to target therapy in a patient in needthereof. An antibody-coated and/or drug-containing nanoparticlecomposition is administered to the patient, then stages of increasingtemperature thermotherapy at the target containing the antibody specificand/or drug-coated nanoparticles are performed. In one embodiment, thetarget is imaged during the temperature increases. The thermotherapy isadministered under conditions (e.g., temperatures, durations, etc.)sufficient to result in targeted therapy in the patient. The inventivemethod limits thermotherapy to a specific target, e.g., a tumor,bacteria, etc. and does not substantially affect normal cells proximatethis target. In one embodiment, the method is performed on a patientreceiving therapy, e.g., chemotherapy, radiation therapy, anti-vascularendothelial growth-factor therapy, and/or steroid therapy. In oneembodiment, the anti-tumor antibody-coated or -containing nanoparticlesfurther comprise a thermosensitive polymer which releases the drugcontained in the nanoparticle by dissociation of the polymer when thethermosensitive temperature is attained. Thermosensitive polymersinclude chitosan-poly(N-isopropylacrylamide), smart polymers,poly(N-isopropylacrylamide (PNIPAM), poloxamers, poloxamines, and/oracid (PMA) polymers modified with thiol groups (PMA_(SH)).

In use, in one embodiment, the composition is administered to thepatient with four subsequent staged increases in temperature. In oneembodiment, the temperature at the target is first increased to resultin drug release from the nanoparticles. These conditions typically occurwith a temperature at the target of at least 35° C. to 43° C., which isprovided for about 0.5 minutes to about 20 minutes. The temperature atthe target is then increased to result in thermotherapy. Theseconditions typically occur with a temperature at the target of 43° C. to50° C., which is provided for about 1 min to about 15 min. Thetemperature at the target is then increased to result in expansion ofthe gases that are dissolved in cellular fluids and concomitantexpansion of the membrane, cytoplasm, and/or nuclear pores. Suchexpansion results in mechanical distension of the cell membrane and/ornuclear membrane with subsequent expansion of the membrane pores and/orchannels allowing essentially unrestricted drug into the cell. Theseconditions typically occur with a temperature from 50° C. to 60° C.,which is provided for about 1 min to about 10 min. The temperature atthe target is then increased to result in water evaporation at thetarget, causing cumulative damage, i.e., essentially unrestricted drugflow combined with protein denaturation and water evaporation. Theseconditions typically occur with a temperature greater than 60° C. (i.e.,a temperature that kills normal cells) and up to 100° C., which isprovided for a duration less than one second to a few minutes.

As one example, in one embodiment, the composition is administered tothe patient, then the temperature increase at the target is limited toless than 60° C. The tumor cells and/or bacterial cells, which arerapidly dividing and hence have increased susceptibility to increasedtemperature, are killed.

Thermotherapy may be provided by, e.g., electromagnetic radiation,ultrasound energy, or an alternating magnetic field. Imaging of thetarget during the temperature increases can control thermotherapy byconnecting the photoacoustic system, which controls the heat generatingsystems, to the electromagnetic radiation-creating instrument, or to areversible magnet, or another ultrasound system generating a focusedultrasound beam. This connection may be, e.g., by a processor. Theoperator instructs the system to achieve the desired temperature, orprovides an algorithm for the system to follow to create the desiredresult, using specified temperatures and exposure durations. Imaging maybe thermal imaging, photoacoustic imaging, X-ray imaging, opticalcoherence tomography, ultrasound imaging, fluorescence imaging,chemiluminescent imaging, positron imaging, surface enhanced Ramanspectroscopy, and/or magnetic resonance imaging (MRI). In oneembodiment, imaging is by MRI.

The composition administered may contain magnetic, diamagnetic,ferromagnetic, and/or paramagnetic nanoparticles. The compositionadministered may contain gold nanoparticles, diamond nanoparticles,platinum nanoparticles, and/or carbon nanoparticle.

The targeted nanoparticles, that contain antibody as well as drug in oron the particles, are administered to a patient in need thereof. Thestaged and selective temperature increases administered at the targetdeliver drug from the nanoparticle to the cell interior, with heatincreasing metabolic need of the target (e.g., tumor cells or bacteria).Thermal therapy is staged as follows: a first temperature at the targetsite of at least 35° C. to 43° C., thereafter a second temperature atthe target site of from 43° C. to 50° C., thereafter a third temperatureat the target site of from 50° C. to 60° C., and thereafter a fourthtemperature at the target site of greater than 60° C. The duration ofthe first stage is from 0.5 min to 20 min. The duration of the secondstage is from 1 min to 15 min. The duration of the third stage is from 1min to 10 min. The duration of the fourth stage is from less than 1second to a few minutes. In embodiments, a stage of thermotherapy can beremoved. For example, the method may be used in a patient with a tumorthat is drug resistant, but the tumor will not be able to developresistance to the increased temperature.

The inventive thermotherapy method results in specific drug delivery andthermal damage to target cells. The method delivers and releases drugfrom the nanoparticles to the cell interior. The increased temperatureincreases the tumor cell/bacteria metabolic needs. Without being boundby a specific theory, the result is cell damage and/or death by drug, byheat, or by the combination of drug and heat.

The staged, selective temperature increase in the nanoparticle can alsoexpand the gas that are dissolved in the cell fluid, creating amechanical stress/damage on the cells/bacteria membrane or internalmetabolic machinery. The internal gas expansion distends the wall of thecell/bacteria and expands the pore size of the cell/bacteria membranes.This in turn causes almost free flow of drug to the cell interior,resulting in cell death. Increasing the temperature, while imaging,beyond 60° C. can also cause not only protein denaturation in the cell,but also cause the water molecules to evaporate. This further increasesthe cell damage leading to cell death. The different stages can becontrolled by the imaging technology described controlling the thermaleffect of the nanoparticles. The processes occur more rapidly and morereadily (faster and sooner) around the nanoparticles than around thesurrounding tissue because of the nanoparticles' small size.

Administration may be by any route and at body site. For example, thecomposition may be administered by an intrathecal, intravenous,intraocular, etc. route. The composition may be administered into a bodycavity (e.g., eye, bladder). The composition may be administered intothe cerebrospinal fluid. The composition may be applied topically, e.g.,to an external or mucosal lesion. In one embodiment, the composition isintravenously administered. The composition may be administered locallyin or near the tumor site, or in or at any body site.

The composition contains a nanoparticle conjugated to an activatablecell penetrating peptide (ACPP), which forms anantitumor-ACPP-nanoparticle-cell complex at the tumor tissue site, ortarget site (e.g., liver). The target site of the patient is exposed toan energy source under conditions sufficient to heat the nanoparticlesin the antitumor-ACPP-nanoparticle-cell complex to a temperature that issufficient to measure an acoustic response produced by the nanoparticle.Exemplary durations are from less than one second to 15 minutes. Thetreatment can be repeated as needed, e.g., within weeks, determined byMRI, CT, ultrasound, PET scans, etc.

In one embodiment, the temperature to measure an acoustic responseproduced by the nanoparticle, indicating damage to the target cells,ranges from about 40° C. to about 60° C. inclusive. A lower thresholdtemperature of 39° C. may be used with drug delivery to break up themedication-nanoparticle complex at the tumor site. In one embodiment,the temperature to measure an acoustic response produced by thenanoparticle, indicating damage to the target cells, ranges from about42° C. to about 45° C. inclusive, or from about 45° C. to about 50° C.inclusive, or from about 50° C. to about 58° C. inclusive. In oneembodiment, for superficially located or otherwise externally accessibletumors, or for treating tumor margins after surgical excision ordebulking, a temperature >58° C. up to 60° C. is acceptable if thetarget is away, e.g. distal or remote, from a vital organ. Suchhyperthermal therapy damages or kills the tumor cells, referred to astumor de-bulking.

In one embodiment of the method, the tumor is surgically excised orremoved prior to or concomitantly with administering the composition andperforming the method. The surgery thus defines the lesion that is atarget area for treatment by the method.

The method may be used on targets or lesions in and/or on any body site.Examples include, but are not limited to, skin, mucosa, organs andtissues of the digestive, genitourinary, nervous (CNS and/or peripheralnerves), respiratory, circulatory, lymphatic, and other systems. As oneexample, a tumor may be located in and/or on a mucosal lining or skin.As one example, cancerous blood cells may be treated by the inventivemethod, e.g., leukemia or lymphoma. In one embodiment, intravenousadministration of the composition permits therapy to circulating bloodor other cells (e.g., immune cells). With surgical tumor excision, thecomposition can be intravenously injected prior to, after, or duringsurgery.

In one embodiment, use of the method to eliminate specific immune cellseffectively reduces or eliminates the need for immunosuppressive drugsin cases of organ transplant. In such an embodiment, nanoparticles arelabeled with specific binding molecules that bind to specific surfacereceptors of these immune activated cells; the method proceeds asdescribed. This embodiment minimizes or eliminates the need to exposethe entire body to potentially damaging radiation, chemotherapy, and/orimmunosuppressant medications, thus minimizing or eliminating theirdamaging side effects.

In one embodiment, use of the method combines hyperthermal treatment, asdisclosed herein, with immunotherapy. The resultant immune-thermaltherapy advantageously uses synergy from the combined approaches,specifically, (i) a patient's inherent response against a non-selfsubstance, in this case a tumor cell, to destroy the tumor cell, and(ii) localized thermal therapy possible by using the inventive methodwith targeted directed nanoparticles.

Thermal therapy, also termed hyperthermal therapy, is completelydescribed, disclosed, and enabled herein. All thermal therapyembodiments may be used in the inventive method.

In embodiments, thermal therapy may be performed simultaneously with, orsubstantially simultaneously with, immunotherapy. In embodiments,thermal therapy may be performed prior to immunotherapy. In embodiments,thermal therapy may be performed subsequent to immunotherapy. Becausethermal therapy is targeted, involves thermal treatment withoutdestroying proteins, and spares non-targeted tissue, it is generallyrelatively safer and less invasive than chemotherapy and/or radiationtherapy.

Targeted therapy includes, but is not limited to, anti-tumor antibodies,other specific binding compounds known in the art such as biotin withstreptavidin, specific receptors and receptor agonists and/orantagonists, etc. Targeted therapy may result when the nanoparticlescontain and/or are coated with one part of the associated pair and thetumor itself and/or an area proximate to the tumor contains thecomplementary part of the associated pair. As another example oftargeted therapy, the nanoparticles may be completely or partially on,associated with, and/or contained in a virus to more specifically targettherapy. As one example and without limitation, the nanoparticle may betargeted to a site of a tumor that has or may have a viral association.Examples include, but are not limited to, papillomaviruses, polyomaviruses, Epstein-Barr virus (EBV), human herpes virus 8 (HHV-8),hepatitis B virus, and human T-cell lymphotrophic virus-1 (HTLV-1). Asanother example, the nanoparticle may be targeted to, incorporated in,or otherwise located at a tumor cell, e.g., membrane viral capsidsand/or envelopes, using a modified-virus and/or adenoma-associated virus(AAV) either alone or in combination with another therapy, e.g., genetherapy. As another example, the nanoparticle may be completely orpartially on, associated with, and/or contained in a tumoricidal virusor a virus rendered tumoricidal to provide additional therapy, e.g., amodified herpes simplex virus selectively targeting malignant cells.

Immunotherapy is broadly defined as therapy to a patient that involvesmodulating at least some part of the immune system, in whole or in part,and whether the immune system involvement stimulates, modulates,suppresses, ameliorates, enhances, treats, etc. the patient. Theimmunotherapy thus may encompass either activation of the patient's ownimmune system or suppression of the patient's own immune system, inwhole or, more likely, in part. Immunotherapy is used to treatneoplastic disease, infectious diseases, inflammatory disease,degenerative disease, etc.

Immunotherapy may provide one or more exogenous compounds that may benatural in whole or in part, recombinant in whole or in part, and/orsynthetic in whole or in part. The type(s) of compounds may be diverse,e.g., small molecules, biologics, enzymes, hormones, protein or peptidefactors, gene modulating agents, etc. Examples include, but are notlimited to, antibodies including polyclonal and monoclonal antibodies,growth factors, integrins, interleukins, cytokines, chemokines,interferons, membrane-coupled receptors, receptor agonists, receptorantagonists, oligonucleotides, mRNA silencers, siRNA, steroids, etc.Upon administration to a patient, an immunotherapy compound acts on thepatient's own immune system in one or more of the ways previouslydescribed, e.g., activation, suppression, enhancement, etc. Theexogenous compound(s) may act directly or indirectly to provide anoutcome. Thus the compound(s) may have a substantially immediate effect,or may have an attenuated or non-immediate effect.

Exemplary immunotherapy compounds include, but are not limited to, thefollowing: polypeptides comprising MHC I and/or MHC II; glycoproteins;APC protein markers; dendritic cell maturation markers; polymers eitherwith or without an lipid group, acrylic polymers, copolymers of lacticacid and glycolic acid; protein, peptide, small molecule, and/orcarbohydrate B cell antigens including toxins; carbohydrate targetingmoieties; lipid targeting moieties; vaccines including the breast cancerHER2/neu peptide, cervical cancer HPV vaccine, colorectal cancer vaccineincluding vaccines against CEA protein, kidney cancer vaccine, lymphomavaccine, lung cancer vaccine include BLP25 (STIMUVAX®), pancreaticcancer vaccine including GVAX and ipilimumab (YERVOY®), prostate cancervaccine including prostate-specific antigen (PSA) and prostate-specificmembrane antigen (PSMA) and PROSTVAC-VF); Toll-Like Receptor (TLR)-1 to10, TLR agonist; toxins; biologics including but not limited to thefollowing: the farnesyltransferase inhibitor tipifarnib; farnesyl andgeranylgeranyl transferase inhibitors; the multikinase inhibitorsorafenib combined with tipifarnib; and bortezomib and tipifarnib.

Exemplary markers to evaluate therapy include, but are not limited to,the following: alpha-fetoprotein (AFP) for liver and germ cell tumors;beta-2-microglobulin (B2M); beta-human chorionic gonadotropin(beta-hCG); BCR-ABL fusion gene; BRAF mutation V600E; CA15-3/CA27.29 inblood for breast cancer; CA19-9 for pancreatic cancer, gallbladdercancer, bile duct cancer, and gastric cancer; BCA-125 in blood forovarian cancer; calcitonin in blood for medullary thyroid cancer;carcinoembryonic antigen (CEA) in blood for colorectal and breastcancer; CD20 in blood for non-Hodgkin lymphoma; chromogranin A (CgA) inblood for neuroendocrine tumors; chromosomes 3, 7, 17, and 9p21 in urinefor bladder cancer; cytokeratin fragments 21-1 in blood for lung cancer;EGFR mutation analysis in tumors (estrogen receptor (ER)/progesteronereceptor (PR)) for non-small cell lung cancer and breast cancer;fibrin/fibrinogen in urine for bladder cancer; HE4 in blood for ovariancancer; HER2/neu in tumor immunoglobulins for breast, gastric, andesophageal cancer, and in blood and urine for multiple myeloma andWaldenstrom macroglobulinemia; KIT in tumors for gastrointestinalstromal tumor and mucosal melanoma; KRAS mutation analysis in tumor forcolorectal and non-small cell lung cancer; lactate dehydrogenase inblood for germ cell tumors; nuclear matrix protein 22 in urine forbladder cancer; prostate-specific antigen (PSA) in blood for prostatecancer; thyroglobulin in tumor for thyroid cancer; urokinase plasminogenactivator (uPA) and plasminogen activator inhibitor (PAI-1) in tumor forbreast cancer; 5-protein signature (Oval) in blood for ovarian cancer;21-gene signature (Oncotype DX) in tumor to evaluate risk of recurrencein breast cancer; 70-gene signature (Mammaprint) in tumor for breastcancer. Generally, a decrease from a patient's previous value and/or anormal level and/or a normal baseline level for the particularindividual indicates therapy efficacy and/or decreased tumor burden.

The effect of any exogenous immunotherapy compound may depend upon itsmethod of delivery which, in turn, may determine how rapidly thecompound is present at the site of action (sometimes termed targetsite). The target site may be one or more types of leukocytes that areclassically regarded as involved with cell-mediated immunity, i.e.,lymphocytes, e.g., T-lymphocytes, B-lymphocytes, or other cell typessuch as macrophages, natural killer cells, mast cells, basophils,dendritic cells, etc. It will be appreciated that many if not all ofthese cell types may contain genetic modifications to affect theirnormal physiological function in the immunologic process. For exampleand without limitation, genetically modified enhancement, decrease,attenuation, facilitation of various targeting and activation functionmay be achieved using techniques known in the art. Because of the myriadtypes of administration routes coupled with the myriad types ofexogenous immunotherapy compounds, delivery may be a consideration inpatient therapy. The exogenous compounds may be delivered in a number ofways. One embodiment provides nanoparticle delivery, with nanoparticlesdescribed herein and as known in the art, and which include but are notlimited to, quantum dots.

As described herein, nanoparticles may be biocompatible or renderedbiocompatible. In one embodiment, nanoparticles may be associated withliposomes. Nanoparticles may contain and/or be coated with, in whole orin part, materials such as ferric oxide, carbon, diamond, zinc oxide,gold, etc. providing methods to target the nanoparticles to a particularcellular, physiologic, and/or anatomic site. For example, one embodimentdescribed herein uses an alternating magnet or electromagnetic radiationwith a metal-containing nanoparticle (e.g., ferromagnetic nanoparticle)to effect therapy (see, e.g., FIG. 5).

Nanoparticles are a particularly advantageous delivery vehicle forseveral reasons. Their small nanoscale size permits their entry,localization, and/or accumulation into anatomical and physiologicalregions otherwise less readily accessible. Their nanoscale size likewisepermits intravenous administration, providing systemic access via thecirculatory system.

Permutations of any of these embodiments are included in the inventivemethod. Illustrative but non-limiting examples include polymer-coatednanoparticles, nanoparticle-stabilized liposome-polymer nanocarriers,nanoparticles contained in or formulated with liposomes, micelles,reverse micelles, etc.

As described herein, the nanoparticles contain a targeting agent, suchas an antibody, a specific binding pair member, etc., ensuring thenanoparticles administered to a patient are directed to a target site,e.g., a tumor site, a particular cellular receptor, etc. Using thisapproach ensures the thermotherapy is targeted.

In one embodiment, a cancer patient receives immunosuppressive therapy.Such immunosuppressive therapy may involve chemotherapy, radiationtherapy, or both chemotherapy and radiation therapy. Prior to thepatient receiving immunotherapy, the patient provides a blood samplefrom which leukocytes are separated. The leukocytes are then cultured invitro in an environment to support their growth and maintenance; T cellsare treated with the cytokine interleukin-2 (IL-2). The culturedautologous leukocytes, termed lymphokine-activated killer cells (LAKcells), are then re-infused into the patient, providing a bolster to thepatient's suppressed immune system. In this embodiment, the combinationof hyperthermal therapy with immunotherapy may be used to advantage.This is because the patient receives combination therapy: (i)chemotherapy and/or radiation therapy, which may or may not be finelytargeted to a specific malignancy, tumor type, tumor location, etc.;(ii) hyperthermal therapy to destroy cells at increased temperature thatdoes not result in protein denaturation thus sparing normal cells andnormal cellular processes, and provided at a specific location to whichthe therapy is directed at a target site; and (iii) immunotherapy tobolster or restore or augment the patient's own immune response and thusbolster or augment natural immune functions. The combination therapyavoids total immunosuppression and provides the patient at least amodicum of immune function during recovery. The combination therapypermits a decreased dose of nanoparticles administered, decreasedfrequency of hyperthermal therapy, or both, providing less of a therapyburden to the patient. The combination therapy, because it considers theimmune system, facilitates patient recovery or comfort with fewercomplications of the immunosuppressive treatment itself.

To effect therapy, one or more large energy generation sources or unitscan provide a source of thermal energy for a large part of the body.Alternatively, a simpler, e.g., smaller, energy generation source orunit, e.g., a hand-held unit, can provide a source of thermal energy tolabeled nanoparticles when brought close to the desired tissue area. Inone embodiment, electromagnetic radiation is the energy source, e.g.,from ultraviolet radiation up to radiofrequency waves. In oneembodiment, ultrasound energy is the energy source, e.g., an ultrasonicunit focusing ultrasound waves to the desired area of the body to heatthe nanoparticles. In one embodiment, an alternating magnetic field isused to heat the magnetic nanoparticles and treat the tumor. Analternating magnetic field is readily created, e.g., by a simple ironrod surrounded by a coil of wire with alternating current.

The device providing the energy source can be a magnet, microwave unit,a radiofrequency (RF) probe, an ultrasound probe, etc. The device can beany size. The device may be stationary, hand held, or introduced intothe body by invasive or minimally invasive methods, e.g., introduced bya catheter. The device can be brought close, adjacent, or proximate tothe target site. If the device is an alternating magnetic field, theoscillation of the magnetic field can range from a few Hz to gigahertzwith appropriate magnetic field force according to the distance from thetarget. In one embodiment, e.g., 0.000001 Tesla to 11 Tesla, with adistance of 0.1 cm to 15 cm, are used.

In one embodiment, non-magnetic, magnetic, or paramagnetic nanoparticleslabeled with the tumor antibody and/or fluorescent moieties are used toselectively bind to tumor cells.

In one embodiment, the method is used to effect therapy of anon-malignant tumor. In one embodiment, the method is used on normalcells with a propensity or predisposition to malfunction and/or growexcessively. One such example includes myofibroblasts, which areactivated after coronary stent placement. Another such example includesnerve cells, which may be aggravated and thus produce chronic pain. Inthese cases, a cell specific antibody is combined with nanoparticles andconjugated ACPPs to attach nanoparticles to axons of these nerves. Thenerves are then treated non-invasively to achieve a localizedtemperature up to about 60° C. to effect selective ablation and, thus,to prevent transmission of pain sensations. Another such example usesthe method therapeutically to stimulate tissue healing, e.g., abeneficial thermal effect in creating heat shock proteins andencouraging circulation in the area.

In one embodiment, after injecting the nanoparticle composition, eitherwith nanoparticles alone or combined with ACPP, the patient target(s) isexposed to an energy source (electromagnetic radiation energy,ultrasound energy, an alternating magnetic field, etc. as previouslydescribed) to increase the temperature of the cells that are complexedwith, i.e., that contain, the nanoparticles. In one embodiment,selection of a desired temperature, e.g., 39° C. to 58° C., duration,and/or imaging means as subsequently described, selectively damages thetumor cells while protecting the normal cells. With surgical tumorexcision, the tumor margins, which contain nanoparticles, are visualizedby imaging means, e.g., photoacoustic imaging, magnetic resonanceimaging, thermal imaging, etc. A magnetic resonance imaging unit can beused for imaging, alone, or for thermal imaging of the tissue.Alternatively, photoacoustic sound can be modified, e.g., amplified, sothe sound can be heard by the surgeon during therapy and be used as adiagnostic test for the presence of the remaining tumor cells in thesurrounding unexcised tissue, guiding further treatment.

The method also permits remote treatment of a patient in need of suchtreatment but unable to travel for on-site treatment by electroniccommunication, e.g., Internet communication. The communication means isoperatively linked to software that controls an energy source and animaging system. The system is integrated to provide an energy source aspreviously described, e.g., ultrasound, electromagnetic radiation,alternating magnetic field, and a thermal imaging system as previouslydescribed, e.g., photoacoustic or MRI. Using this embodiment, medicalpersonnel provide instructions that control both the imaging device andthe heating device. When a desired temperature in the target tissue isachieved, the software modifies the thermal energy output, e.g., itmaintains the tissue temperature at the desired thermal energy level orit increases or decreases the thermal energy at its source. In oneembodiment, the system is equipped with a tracking device and patternrecognition software to maintain an accurate location of the treatmentsite. In one embodiment, any variation, such as patient movement or achange in settings, triggers a failsafe system to terminate theprocedure. In one embodiment, the entire system provides real-timeresults to medical personnel at a location remote from the patient(e.g., down the hall or in a different country), and these medicalpersonnel can control the entire process electronically by Internetconnection. The information is software controlled, medical personnelregulated, and subject to the above-described failsafe mechanisms andprivacy standards as needed.

The target site is heated to the desired temperature to hyperthermallytreat the target site using standard heating instruments and equipmentfor heating tissue and standard equipment for visualizing the dye in thetarget site that has been released from the heat sensitive particles.For example, the heating equipment preferably includes suitable heat orenergy source that is able to focus the heat or energy on the target andis able to control heat and temperature of the tissue. The heat sourcecan be an electrical resistance heating element, or an indirectly heatedelement. The heating device can also have a radiation energy source forproducing heat at the target site, such as a radio frequency (RF)device, ultrasonic generators, laser, or infrared device. One example ofan RF generator device for hyperthermally treating tissue in a selectedtarget site is disclosed in U.S. Pat. No. 6,197,022, which is herebyincorporated by reference in its entirety. Examples of suitableultrasonic devices for delivering ultrasonic hyperthermia are disclosedin U.S. Pat. Nos. 4,620,546, 4,658,828 and 4,586,512, the disclosures ofwhich are hereby incorporated by reference in their entirety.

In one embodiment, the duration of the applied radiation energy may befrom about a femtosecond to about 15 minutes. In one embodiment, theduration of the applied radiation energy may be from about onepicosecond to about 15 minutes. In one embodiment, the duration of theapplied radiation energy may be from about one nanosecond to about 15minutes. In one embodiment, the duration of the applied radiation energymay be from about one microsecond to about 15 minutes. In oneembodiment, the duration of the applied radiation energy may be fromabout 1 second to about 15 minutes. In one embodiment, the duration ofthe applied radiation energy may be from about 1 second to about 15seconds.

In one embodiment, the temperature at which the radiation energy isapplied ranges between about 45° C. to about 49° C. In one embodiment,the temperature at which the radiation energy is applied ranges betweenabout 39° C. to about 58° C. In one embodiment, the temperature at whichthe radiation energy is applied ranges between about 39° C. to about 45°C. In one embodiment, the temperature at which the radiation energy isapplied ranges between about 45° C. to about 49° C. In one embodiment,the temperature at which the radiation energy is applied ranges betweenabout 49° C. to about 56° C. For example and without limitation,relatively brief treatment times are used for circulating cells (e.g.,cells in vessels of the circulatory and lymphatic systems). Relativelylonger laser pulses may also be used for tissues located deep inside thebody. In one embodiment, the ultrasound frequency can range between 0.5KHz to 200 MHz. In one embodiment, the ultrasound frequency can rangebetween 0.5 M Hz to 10 MHz. In one embodiment, the ultrasound frequencycan range between 10 MHz to 30 MHz. In one embodiment, the ultrasoundfrequency can range between 5 MHz to 80 MHz.

In one embodiment, the heat source includes a probe having a tip withthe heating element or energy emitting element attached thereto. Theenergy emitting element can be an optical fiber operatively connected toa laser, infrared or ultraviolet light source. The probe preferablyincludes a suitable control mechanism for manipulating the probe to thetarget site and a control for controlling the energy applied to thetarget site. In one embodiment the wavelength of light is selected to bein the range between 350 nm to 1300 nm. In another embodiment thewavelength of light is selected to be in the range between 450 nm to 600nm.

A suitable device for hyperthermally treating the tissue in a targetsite is shown in the figure. The device 10 includes a probe 12 having anoptical fiber 14 with a distal end 16 for emitting a laser light to heatthe tissue 17. Preferably, the end 16 of optical fiber 14 can focus thelight source on the target site 17. Optical fiber 14 is connected tolaser generator 18 that is able to generate a laser beam of sufficientintensity and within wavelength for hyperthermally treating tissue. Foruse of the method in making a diagnostic assessment or for therapy, thetissue is treated to a temperature of at least 41° C. to 56 C.°, andpreferably at least 42° C. to 56° C. In a preferred embodiment, probe 12includes a second optical fiber 20 having a distal end 22 and a thirdoptical fiber 24 having a distal end 26. Optical fiber 20 is operativelyconnected to a light source 28, such as a laser, that is able to emit alight beam having a wavelength capable of fluorescing a fluorescent dyein the target area when the dye is released from the heat sensitiveparticles. Optical fiber 24 is operatively connected to a suitableimaging device 30 for capturing the fluoresced light from the exciteddye and visualizing and producing an image of the fluorescing dye in thetarget site. Imaging device 30 can be a CCD or a device equivalent to afunduscope. An example of a suitable funduscope is disclosed in U.S.Pat. No. 4,891,043 to Zeimer, which is hereby incorporated by referencein its entirety.

In another embodiment of the invention, the probe can include a heatingelement or a device for receiving a heated fluid that can transfer theheat to the tissue in the target site. The probe can include anexpandable bladder member for receiving a heated fluid delivered from afluid-heating source. In still another embodiment, the expandablebladder includes a permeable portion so that the heated fluid can beapplied directly to the target site. A suitable aspirating device ispreferably included to remove the excess heating fluid when applieddirectly to the target site.

In one embodiment, the target site is the retina or choroid in the eyeof the patient. The heating and visualizing instrument includes a lasercapable of focusing a laser beam on the target site where the laser beamhas a wavelength and intensity to heat the cells to a temperature of atleast 42° C. In one embodiment, the laser heats the cells to atemperature of 50° C. or below and preferably to about 42° C. to 56° C.The instrument also includes or is used in combination with a funduscopeto excite or fluoresce the dye that has been released in the target siteto capture and visualize the fluorescing dye. A funduscope that can beused is disclosed in U.S. Pat. No. 6,248,727, which is herebyincorporated by reference in its entirety. The laser source is selectedto provide sufficient energy to heat the tissue in the target site tothe desired temperature.

The fluorescent dye is encapsulated in a suitable heat sensitiveparticle and introduced into the patient in a location to be visualizedin the target site. The heat sensitive particles can be microcapsules,or nanocapsules that are able to release the dye at a temperature ofabout 41° C., and preferably 42° C. or higher. In preferred embodiments,the fluorescent dyes are incorporated into heat sensitive liposomes thathave a phase transition temperature at the temperature of hyperthermia.In one embodiment, the liposomes have a phase transition temperaturewithin the desired temperature range that tissue or cells are to beheated.

In one embodiment, the liposomes have a phase transition temperature ofat least 41° C. and preferably at least 42° C. In a preferredembodiment, the liposomes have a phase transition temperature of about45° C. to about 50° C.

The liposomes can be made by various processes as known in the art. Thephase transition temperature of the phospholipid is selected to controlthe temperature that the dye and other components are released from theliposomes. Phospholipids are known to have different phase transitiontemperatures and can be used to produce liposomes having releasetemperatures corresponding to the phase transition of the phospholipids.Suitable phospholipids include, for example, dimyristoylphosphatidylcholine having a phase transition temperature of 23.9° C.,palmitoylmyristoylphosphatidyl choline having a phase transitiontemperature of 27.2° C., myristolypalmitoylphosphatidyl choline having aphase transition temperature of 35.3° C., dipalmitoylphosphatidylcholine having a phase transition temperature of 41.4° C.,stearoylpalmitoylphosphatidyl choline having a phase transitiontemperature of 44.0° C., palmitoylstearolyphosphatidyl choline having aphase transition of 47.4° C., and distearolyphosphatidyl choline havinga phase transition temperature of 54.9° C. Another suitable phospholipidis a synthetic C₁₇ phosphatidyl choline from Aventi Inc. having a phasetransition temperature of about 48° C.-49° C.

The phase transition temperature and the release temperature of theliposomes can be selected by combining the different phospholipidsduring the production of the liposomes according to the respective phasetransition temperature. The phase transition of the resulting liposomemembrane is generally proportional to the ratio by weight of theindividual phospholipids. Thus, the composition of the phospholipids areselected based on the respective phase transition temperature so thatthe phase transition temperature of the liposome membrane will fallwithin the selected range. By adjusting the phase transition temperatureof the liposome membrane to the selected range, the temperature at whichthe liposomes release the dyes and other components can be controlledduring hyperthermia.

The liposomes in one embodiment of the invention are preferably preparedso that the liposome membrane has a phase transition temperature of atleast 42° C., and preferably about 42° C. to about 50° C. In a preferredembodiment, the liposomes leak or rupture at a temperature of about 49°C. or less, and typically between about 45° C. and 49° C. In oneembodiment, the phospholipids have saturated acyl groups. For example,glycerophospholipids can be used that have two acyl groups having 8 ormore carbon atoms and where at least one of the acyl groups have atleast 10 carbon atoms and typically 12-18 carbon atoms. Examples ofsuitable phospholipids include hydrogenated lecithin from plants andanimals, such as egg yolk lecithin and soybean lecithin. Thephospholipid can also be phosphatidyl choline produced from partial orcomplete synthesis containing mixed acyl groups of lauryl, myristoyl,palmitoyl and stearoyl.

The liposomes can be prepared from a mixture of dipalmitoylphosphatidylcholine and disteroylphosphatidyl choline in a weight ratio of 95:5 toabout 5:95 and generally about 80:20 to about 20:80. In one embodiment,the liposomes are made from a mixture of dipalmitoylphosphatidyl cholineand disteroylphosphatidyl choline in a ratio of 45:55 to about 55:45provide a phase transition temperature of about 46° C. to about 49° C.

The liposomes of the invention can be prepared by standard processes asknown in the art. The liposomes can be unilamellar or multilamellar andhave a particle suitable for delivering the dye to the target site. Inone embodiment, the liposomes have a particle size of a sufficientlysmall size to be introduced into the bloodstream of the patient in alocation near the target site to flow through the target site.

The liposomes can contain a suitable osmotic pressure controlling agentthat is physiologically acceptable to the patient. Examples includesodium chloride, sugars such as glucose, mannitol and sorbitol, andamino acids such as glycine, aspartic acid and glutamic acid. Examplesof suitable process for preparing liposomes are disclosed in U.S. Pat.No. 4,235,871 to Papahadjopoulos et al. and U.S. Pat. No. 4,522,803 toLenk, which are hereby incorporated by reference in their entirety.

The liposomes of the invention contain a dye that is able to fluoresceand that can be visualized in the target site by exciting with a lightsource that is amenable to the target site and the patient. Thefluorescent dye can be any fluorescent that is suitable forencapsulation and is physiologically compatible. Preferably, thefluorescent dye is quenched when encapsulated at an appropriateconcentration. The quenching concentration is a sufficiently highconcentration to mask or minimize detection of fluorescence whenilluminated by a fluorescing light source. The quenching concentrationcan be determined by routine experimentation as known in the art. Whenheated, the heat sensitive liposomes rupture or leak the dye and othercomponents of the liposome so that the dye is diluted in the target siteto a suitable concentration where the dye can be fluoresced andvisualized upon excitation by a suitable light source. Examples ofsuitable dyes include 6-carboxyfluorescein and its derivatives. Suitablefluorescent dyes can be excited by an emit light at wavelengths that arenot strongly absorbed by the tissue. Other suitable dyes includeindocyanin green and aluminum phthalocyaninetetrasulfonate. It will beunderstood that the fluorescing light source and the visualizinginstrument are selected according to the wavelength of the fluorescingdye to visualize the dye.

In one embodiment, the dye is selected to fluoresce in the presence of alight from an argon laser, a helium-neon laser or infrared laser.Preferably the dye is selected to be compatible with the exciting lightor laser source to fluoresce when subjected to the light or laser beam.A suitable dye is sold under the tradename D-275 by Molecular Probes,Inc. and fluoresces green when exposed to light from an argon laser at484 nm. A dye sold under the tradename D-1121 fluoresces orange whenexposed to a long wavelength laser light at 560-574 nm. One preferreddye is an infrared excitable dye Dilc₁₈(7), which fluoresces at awavelength of 740-780 nm.

In one embodiment of the invention, a fluorescent dye is encapsulated inliposomes having a phase transition temperature of 42° C. to 50° C., andpreferably about 45° C. to 49° C. In another embodiment, the liposomeshave a phase transition temperature to release the dye at a temperatureof about 46° C. to about 49° C. The liposomes are injected into thebloodstream of the patient in a location where the liposomes flow to thetarget site. In some embodiments, the liposomes can be introduceddirectly to the target site intravenously, subcutaneously or topically.A hyperthermal heat source and a dye exciting light source are appliedto the target site. The hyperthermal heat source, which is preferably alaser light beam, is focused on the target site to heat the tissue andthe cells to a temperature of at least 42° C. to hyperthermally treatthe tissue and kill the cells. The hyperthermal heat source also heatsthe liposomes to a temperature at least equal to the phase transitiontemperature to release the dye. The fluorescing light source excites thedye so that the fluorescing dye in the target site can be detected andvisualized. By encapsulating the fluorescent dye in liposomes having aphase transition of at least 42° C., the detection of the fluorescingdye provides a positive indication to the operator that the desiredtissue temperature has been obtained that is necessary to hyperthermallytreat the tissue. The phase transition temperature of the liposomes isselected according to desired minimum temperature that the tissue is tobe heated. The hyperthermia energy source is applied to the target sitefor a time sufficient to treat the tissue to the desired level.Generally, the tissue is heated to a temperature of at least 42° C. for1-15 minutes and preferably 1-10 minutes.

In one preferred embodiment of the invention, the liposomes contain asuitable drug or photosensitizing agent. The drugs preferably show asynergistic effect when combined with the hyperthermia treatment of theinvention. The release of the drugs from the liposomes provide animproved targeting effect by releasing the drugs by the heat source inthe target site. Suitable drugs include antitumor agents such ascisplatin, carboplatin, tetraplatin and iproplatin. Suitable anticancerdrugs include adriamycin, mitomycin C, actinomycin, ansamitocin and itsderivatives, bleomycin, Ara-C, daunomycin, metabolic antagonists such as5-FU, methotrexate, isobutyl5-fluoro-6-E-furfurylideneamino-xy-1,2,3,4,5,6hexahydro-2,4-dioxopyrimidine-5-carboxylate. Other antitumor agentsinclude melpharan, mitoxantrone and lymphokines. Antitumor therapyinvolving biologics, e.g., DNA, RNA, protein, siRNA, genes or portionsthereof, etc., may be administered.

The amount of the particular drug entrapped in the liposomes areselected according to the desired therapeutic dose and the unit dose.

In one embodiment, the method administers a drug or drugs and/or aradiotracer, either liposome-contained or not, contained innanoparticles. The nanoparticles are formulated, using methods known byone skilled in the art, to release the drug and/or radiotracer when acertain temperature is attained. As only one example, the nanoparticlescan be conjugated with thermosensitive polymers that will disassociateand hence will release the drug and/or radiotracer upon reaching andexceeding a given temperature.

Such thermosensitive polymers and methods of formulating thesethermosensitive polymers with or in nanoparticles containingchemotherapy drugs and/or a radiotracer are known in the art.Embodiments of the disclosed method use combinations of thermosensitivepolymers with, e.g., known pH sensitive polymers, magneticallycontrolled release agents, magnetoliposome agents, thermosensitiveliposomes entrapping iron oxide nanoparticles, etc., to achievecontrolled drug release. The following non-limited examples areexpressly incorporated by reference herein and generally describe suchpolymers and methods. Alavarez-Lorenzo et al. describetemperature-sensitive chitosan-poly(N-isopropylacrylamide)interpenetrated networks that have enhanced loading capacity andcontrolled release properties. Alexander et al. describe variablearchitecture polymers that are temperature- and pH-responsive, termedsmart polymers, including synthetic copolymers and polymer derivatives.Hoare et al. describe nanoparticles encased inpoly(N-isopropylacrylamide) (PNIPAM) that are heated by magneticinduction. Mornet et al. disclose poloxamers and poloxamines that aresuitable vehicles acid (PMA) polymers that are modified with thiolgroups (PMA_(SH)) and are suitable vehicles for delivery of thenanoparticles and anticancer drug(s). In one embodiment, thenanoparticles are conjugated with radiological imaging compounds, e.g.,gadolinium used in magnetic resonance imaging. In one embodiment, thenanoparticles are conjugated with metabolically active compounds, e.g.,F-18FDG used to visualize tumors and metastatic lesions using positronemission tomography-computed tomography (PET-CT) scanning. It is knownthat heating cells to a temperature greater than 60° C. denatures itsproteins and/or coagulates its contents, but due to the specificity ofthe antibody targeting, only the tumor cells are thermally treated;normal cells are unperturbed. Thus, these methods are used either singlyor in combination to achieve desired effects of targeting tumor cells,and specifically tumor stem cells that are otherwise resistant totreatment and/or that elude detection.

The specific tissue site to be thermally treated, containing the complexof polymer-coated drug-containing nanoparticle and antibody complex, isthermally treated by any appropriate modality (e.g., electromagneticradiation, ultrasonic radiation, alternating magnetic field, etc.) to afirst temperature at which the polymer and/or liposome releases drug. Inone embodiment, this temperature is achieved relatively early in thermaltreatment, e.g., from 37° C. to 43° C. After release, the nanoparticlesare further thermally treated to achieve or exceed a second temperature,e.g., from 43° C. to 45° C., 43° C. to 58° C., 45° C. to 50° C., 50° C.to 58° C., 58° C. to 60° C., to greater than 58° C., or to greater than60° C. Without being bound by a single theory, the first thermaltreatment primes the tumor cells and the tumor stem cells. When thepatient is receiving or has received chemotherapy, the priming resultsin the tumor cells' enhanced susceptibility to both the effect ofchemotherapeutic drug and the damage from the rise of thermal energyinside the tumor mass. When the patient is receiving thermal therapysingly, or before chemotherapy, the priming results in the tumor cells'enhanced susceptibility to the damage from the rise of thermal energyinside the tumor mass, then are further damaged upon administration ofhigh doses of chemotherapy, either locally administered to target to thetumor site. At the relatively high temperatures at or exceeding 43° C.,thermal damage is relatively selective in that it occurs primarily tocells with higher metabolisms, i.e., tumor cells. The method thus leavesnon-tumor cells unperturbed or minimally perturbed by the treatment.

Examples of suitable photosensitive (photosensitizer) agents includeaminolevulinic acid, porphyrin derivatives, porpurine derivatives,NPE-6, ATX-10, plant derived photosensitizers. Other syntheticsensitizers such as SNET₂ and Lutex can be used. Preferably, thephotosensitizers are used in non-toxic amounts. In other embodiments,the liposome compositions can contain liposomes that encapsulate ahyperthermic potentiating agent such as perfluorooctyliodide,perfluorotributylamine, perfluorotripropylamine, andperfluorooctylbromide. Examples of liposome encapsulated potentiatorsare disclosed in U.S. Pat. No. 5,149,319 to Unger, which is herebyincorporated by reference in its entirety. Other bioactive agents thatcan be delivered to the target site by encapsulating in liposomesinclude anti-inflammatory agents, antibiotics, antibacterial agents,antifungal agents, anti-neoplastic agents and antiparasitic agents.Examples of anti-neoplastic agents include aclacinomycins, chromycinsand olivomycins.

In another embodiment of the invention, the liposome compositioncontains a mixture of liposomes having different phase transitiontemperature to release the components at different temperatures. In oneembodiment, the liposome composition contains liposomes encapsulating afirst dye and having a phase transition temperature of 42° C. to about45° C. and liposomes encapsulating a second dye and having a phasetransition temperature of about 50° C. or higher. In one embodiment, thesecond dye is encapsulated in liposome that release the dye at atemperature range of 50° C. to 60° C. Preferably, the second dye is ableto fluoresce at different color than the first dye so that the dyes aredistinguishable. In this embodiment, the liposome composition isdelivered to the target and the target site is subjected to hyperthermiatemperatures. As the tissue in the target site is heated to at least 42°C., the first liposomes rupture or release the first dye so that thefirst dye is visualized and detected in the target site. The detectionof the first dye in the target site enables the operator to monitor thetemperature of the tissue in the target site and to indicate that ahyperthermal temperature has been attained in the tissue at the targetsite. During hyperthermia, it is difficult to determine and monitor theactual temperature of the tissue and care must be taken to avoidoverheating of the tissue and denaturization of the proteins. Inpreferred embodiments of the invention, the hyperthermal treatment doesnot exceed the protein denaturization temperature. In this embodiment,the second liposomes are selected to rupture or release the second dyeat or slightly below the protein denaturization temperature. In thismanner, the second dye is released and visualized to provide theoperator with an indication that the tissue is heated to the proteindenaturization temperature. The heat source is then adjusted by theoperator to reduce the energy applied to the target site to avoidprotein denaturization.

In another embodiment, the liposome composition can contain severalliposomes that can leak or rupture at different temperatures to releasethe dyes at incremental temperatures as the temperature of the targetsite increases. In one embodiment, the liposomes can be selected to leakor rupture the dye at 2° C. intervals between about 42° C. and 50° C.The dyes for each liposome can be different to fluoresce a differentcolor so that the different colors indicate a different temperature ofthe target site. In other embodiments of the invention, the tissue inthe target site can be irradiated by beta radiation from strontium oriridium isotopes. Gamma radiation from P³², iodine-95, and palladium-90can also be used. The radioactive isotopes can be delivered as smallparticles to the target site in combination with the hyperthermiatreatment.

In another embodiment, the method combines thermal therapy, either withor without chemotherapy, with localized internal ionizing radiationtherapy to treat a patient in need of such treatment. The conditions ofthermal therapy are described herein, and chemotherapy is also describedherein and is known by those skilled in this art. The addition oflocalized internal ionization radiation therapy damage or destroy theendothelial cells lining the vessels proximate the tumor site, in effectstarving the tumor of its nutrient supply source. This embodiment of themethod administers a composition of targeted nanoparticles thatcontaining an anti-tumor antibody, a radioactive isotope, and may alsocontain drug, to a patient. The patient receives an internal dose ofradiation from the administered radioactive nanoparticles, i.e.,internal radiation therapy, in contrast to external radiation therapywhere the patient receives radiation from an external radiation source.This combination of localized thermotherapy and localized internalradiation therapy, and optionally localized chemotherapy, i.e., drugtherapy, increases the likelihood of eradicating both existing tumorcells and tumor cell progenitors, as well as damaging endothelial cellsin adjacent tumor-associated vessels. The method thus results in morerobust therapy and increased chances of patient survival.

This embodiment relies on thermotherapy provided at the target site, andinternal radiation therapy at the target site. In general, and assubsequently described, a composition of anti-tumor cell antibodyassociated with radioactive nanoparticles is administered to thepatient, e.g., by intravenous injection. The tumor cell specificity ofthe antibody localizes the composition at the tumor site forthermotherapy, i.e., controlled hyperthermal therapy, and the associatedradioactive nanoparticles damage or destroy the vessels adjacent thetumor, e.g., blood vessels, lymphatic vessels.

Thermotherapy is administered under conditions sufficient to result intargeted therapy to cells in the patient, as described herein. Radiationtherapy results in damage to the tumor cells and adjacent endothelialcells in vessels providing blood, lymph, etc. to the tumor, i.e., tovessels feeding the tumor. As one example, nanoparticles can be coatedwith antitumor antibody to target, e.g., glioma cells in a patient witha brain tumor; an antibacterial antibody and an appropriate antibiotic,etc. Nanoparticles may be conjugated either directly with radioisotopes,or radioisotopes may be conjugated with polymers that are subsequentlyused coat the nanoparticles. Monoclonal antibodies may be used to attachcoated nanoparticles to tumor cell surface markers, bacterial cellsurface markers, etc. Nanoparticles are then provided, e.g.,intravenously injected using a needle or catheter, applied through anexternal orifice, to target the desired location. The dose can vary,e.g., from 10 μCi to 20,000 μCi. Radioisotopes producing α radiation arepreferred, examples of which are At²¹¹ (α particle, duration 7.2 hr,penetration 0.08); Ac²²⁵ (α, β particle, duration 10 days, penetration10 0.1 mm); Bi²¹² (a, particle, duration 60.6 min, penetration 0.09);Bi²¹³ (α, β particle, duration 46 min, penetration >0.1 mm); Ra²²³ (α, βparticle, duration 11.4 days, penetration >0.1 mm); Pb²¹² (α, βparticle, duration 10.6 hr, penetration >0.1 mm); Tb¹⁴⁹ (α, β particle,duration 4.2 hr, penetration >0.1 mm); I¹³¹ (α, λ particle, duration 193hr, penetration 2.0 mm); Cu⁶⁴ (α, λ particle, duration 193 hr,penetration 2.0 mm); I¹³¹ (α, λ particle, duration 193 hr, penetration2.0 mm); and numerous other a emitters that can be similarly used, e.g.,Bi²¹³, Bi²¹², the boron neutron capture therapy (BNCT) that can be usedfor superficial radiation penetration of tumor cells or adjacent cellsto a depth of 7 μm. Similarly, groups of non-magnetic, paramagnetic, ormagnetic nanoparticles can be produced. Polymeric coating of thesenanoparticles renders them less toxic to normal tissue. Iodine¹³¹ may becrosslinked with antibody-coated nanoparticles to label the tumor orbacterial cells, both for therapy and imaging. Anti-integrins oranti-VEGFs may also be incorporated. The nanoparticles may be usedsimultaneously for drug delivery purposes against these tumor orbacterial cells, with the drug in PDA, PGLA, dextran, dendrimers, PEG,etc. The nanoparticles may be quantum dots, polymer based nanoparticles,colloidal gold nanoparticles, iron oxide nanoparticles, etc. Thenanoparticle size may range from 2 nm to 400 nm in one embodiment, andfrom 5 nm to 200 nm in another embodiment.

Alpha-emitting isotopes emit radiation for a relatively short time, thustheir effect will be short lasting and not damage normal tissue adjacentto a tumor. Isotopes of a radiation emitted penetrate the tissue for arelatively short distance. Because the nanoparticles are attracted tothe tumor cell membranes, radiation affects not only the tumor cell butalso the adjacent faxcular endothelial cells. The endothelial cells arespecifically very sensitive to the radiation, therefore theirobliteration deprives the tumor cells of their nutrient source.

This treatment combination disrupts the vessels, to narrow the vessellumen and/or damage the vessels themselves, killing both the tumor cellsby the controlled thermotherapy with optional chemotherapy, anddisrupting the nutrient source for the tumor and progenitor stem cells.

In one use of this embodiment of combined thermotherapy andradiotherapy, vessels are effected such that the neovascularization inwet age related macular degeneration (wet AMD) is treated. To treat AMD,local choroidal injection is used. The dose can vary depending on themode of administration, body mass, body size, location of the tumor,etc. A does may range from 10 μCi to 20,000 μCi.

The radioactive nanoparticles for localized internal radiation therapymay be ferromagnetic or nonferromagnetic, as described herein. They maybe coated and/or otherwise conjugated, as described herein, with one ormore anti-tumor cell antibodies, as described herein, then intravenouslyinjected into or otherwise administered to the patient. The antibodyconjugated and/or coated radioactive nanoparticles then selectively bindto the targeted tumor cells, with the nanoparticles emitting eitheralpha radiation, gamma radiation, or beta radiation within a localizeddiameter of 1 mm to 2 mm from the site. The 1 mm to 2 mm radiationpenetration range self-limits the radiation effect to thetumor-localized site, while desirably damaging endothelial cellsadjacent the tumor. The radiation range damages endothelial cells invessels feeding the tumor, diminishing or obliterating its nutrientsource and in effect starving the tumor cells, as well as damaging tumorprogenitor cells. The method targets all sites of tumor cells, andtherefore treats both the primary lesion site and any metastatic sites.

Thermotherapy in combination with radiotherapy is performed using acontrolled energy source, e.g., computer controlled, as describedherein. A local magnetic and/or electromagnetic field is applied andmonitored as described herein, e.g., MRI or photoacoustic imaging. Inthis way, the targeted cells and associated and proximate vasculatureare affected. Non-targeted cells and distal vasculature, i.e., more than1 mm to 2 mm from the tumor localized site, are not affected.

Another embodiment of the invention images the heat (temperature)production inside the eye (target) tissue. The desired temperature isachieved using a laser and photoacoustic imaging technique.

It is known that light can generate sound waves. This is the basis ofphotoacoustic technology. Some of the delivered energy, e.g., by laser,will be absorbed and converted into heat, leading to transientthermoelastic expansion and thus ultrasonic emission. The generatedultrasonic waves are then detected by ultrasonic transducers to formimages. It is known that optical absorption is closely associated withphysiologic properties, such as hemoglobin concentration and oxygensaturation. As a result, the magnitude of the ultrasonic emission, i.e.,the photoacoustic signal, that is proportional to the local energydeposition, reveals physiological specific optical absorption contrast.Two- or three-dimensional images of the targeted tissues can then beformed.

A photoacoustic image is independently generated from an ultrasonicimage, however, it is only visualized, i.e., imaged, by an ultrasonicreceiver. Therefore, photoacoustic imaging and ultrasonic imaging shouldbe performed simultaneously, so that the additional changes in theultrasonic image, resulting from heat expansion, can be seen. In fact,if the ultrasonic wave and photoacoustic wave are parallel, there is noneed for any other visualization system, as is the case is opaquetissue. The changes in the ultrasonic images are, however, dependent onthe temperature that is created by an additional electromagneticradiation, such as light, microwave generator, etc. This may be usedwhen microwaves are used to treat intraocular tumors.

Presently, acoustically coupled resonant optical systems are sensitiveenough to detect sound waves and differentiate the sound waves based onthe temperature generated inside the target tissue. The pulsed lightpropagates in the ocular tissue uninterrupted until it meets theretino-choroidal tissue where it is differentially absorbed by thetissue producing a spatial distribution of the sound sources that can beimaged by an array of acoustic sensors. All visible light wavelengthsand infrared wavelengths up to 1300 nm pass through the eye easilyreaching the retina and choroids thus creating a high contrast betweenthese tissue components. In the eye, most of retinal-choroidal pathologyrelates to the abnormal vessel formation, located at the junction of theretina and the choroids (new vessels), and most of the intraoculartumors are of retinal or choroidal origin permitting light, specificallyinfrared light at a wavelength of about 780 nm to about 1300 nm topenetrate these structures. The use of incoherent or partially coherentlight permits penetration of a few centimeter and maintains a goodspatial resolution for diagnosis or treatment. The ocular tissue, fromthe cornea to the retina, provide a uniform optical density and index ofrefraction. This index of refraction changes when the light reaches theretina and choroids. However, the contrast image, in ultrasonic imaging,is related to the density and compressibility of the tissue, not theindex of refraction, thus permitting photoacoustic imaging to be used inevaluating functional properties of certain molecules based on differentoptical absorption of molecules, e.g. in oxymetry differentiatingoxygenated and reduced hemoglobin.

The diagnostic application of photoacoustic imaging is based on theabsorption of electromagnetic energy by different molecules, producingdifferent changes in temperature, pressure, and density. Therefore,photoacoustic image generation is the result of photothermal effect onthe tissue or molecules.

If the laser pulse is short enough, a local acoustic effect is generatedthat can be imaged by an ultrasonic transducer in 2D or 3D format.Because photoacoustic and ultrasonic imaging can share the same arrayand receiver, the image produced by them can simultaneously provideinformation on the thermal and anatomical structure, and location of thetissue in a rapid succession such as real time (video) images.

Nanosecond pulses can be generated from a Nd-YAG or Alexandrite laser.The laser delivery can be done either as a combined transducer-laserhead or independently through any optical system such as a slit lamp, adirect or indirect ophthalmoscope, or a fundus camera. These instrumentshave their independent illuminations permitting simultaneousvisualization or imaging of the lesion in the eye using multiple imagingmodalities, potentially along the previously described markers such asliposomes. In this case, the ultrasonic images are obtained through anindependent transducer.

A contrast agent or a marker (biomarker) can be used to enhance theimage or temperature (heat production) in the growing cells, such aschoroidal neovascularization or tumor cells. For example, goldnanoparticles or tubes can be injected systemically which has a lightabsorption around 800 nm wavelength, which corresponds to a laser oftenused in ophthalmology for retinal coagulation purposes. The contrastgenerated by the above biomarkers in photoacoustic imaging isproportional to the concentration of the biomarker. The goldnanoparticles may be any shape, e.g., spherical, ellipsoidal, tubular(cylindrical). The gold nanoparticles may be solid or hollow. The sizeof the gold nanoparticles may range from 2 nm to 700 nm. In oneembodiment, the size of the gold nanoparticles ranges from 50 nm to 250nm.

In one embodiment, the thermal images, generated using photoacousticimaging, can indicate progressively increasing tissue temperature whilethe area is being treated. In one embodiment, a laser is used to treatthe area while photoacoustic imaging is used to generate thermal imagesof the treatment area. In one embodiment, studies can be conducted todemonstrate the relationship between the photoacoustic images generated,as a result of a certain energy input, and incremental temperature risein the tissue to create a target temperature, for example, up to 55° C.,or any other temperature below the temperature of protein denaturation.

Upon injection of labeled gold nanoparticles, their concentration in theperipheral blood increases. The concentration is reduced within about 24hours, depending in part on whether the gold nanoparticles contain(poly)ethylene glycol (PEG) groups or not (i.e., are PEGylated or not),because of hepatic clearance. The labeled gold nanoparticles areprimarily either present in a bound form, e.g., anantibody-labeled-nanoparticle-cell (malignant cell) complex, or areabsorbed by the tumor cells and hence are internalized. Hence, theirdecreased concentration in peripheral blood (e.g., blood sample obtainedby venipuncture) indicates presence of a tumor cell, and providesquantitative information about the number of labeled cells per ml blood.Subsequently the total number of circulating cells in the total wholeblood volume of a patient can be calculated. This permits discovery thepresence of malignant cells, the ability to quantify the amount ofcirculating malignant cells from one lesion or multiple metastaticlesions which have metastasized, and to obtain information on theirlocations in the body.

Obtaining blood samples over a period of time (hours, days, weeks, etc.)and subsequently measuring the concentration of the labeled particles,indicates an increase or decrease in tumor cell shedding as a result ofany therapy such as radiation, chemotherapy, thermotherapy, etc. It isthus of diagnostic usefulness, e.g., to monitor efficacy of therapy in apatient receiving therapy for a tumor. The patient receives anintravenous injection of a defined concentration of labeled goldnanoparticles. The concentration of the labeled nanoparticles in theperipheral blood is determined; this is a base level. Then, at a definedinterval (e.g., days, weeks, etc.), as determined by a healthprofessional, another peripheral blood concentration of the labelednanoparticles is determined as an indicator of labeled nanoparticleclearance from the peripheral blood. The decrease in nanoparticleconcentration is determined, and correlated with the previous uptake todetermine the presence of a tumor, absence of a tumor, or decreasedtumor burden (shedding). To illustrate, e.g., a decrease up to 30% toover 95% in 7 days indicates normal clearance of the labelednanoparticles, and a decrease up to 99% over 7 days to 14 days indicatesinternalization of the labeled nanoparticles by a tumor. Any significantdetection of nanoparticles beyond this time period indicates presence ofcirculating tumor cells; confirmation is obtained by evaluating aperipheral blood sample obtained by venipuncture and/or by tumor cellhistological examination.

In embodiments, the gold nanoparticles can be injected into a tumor,injected into a body cavity, applied over a mucosal surface, and/orapplied to skin having a tumor. The absorption of the labeled particlescan be used to differentiate a benign tumor from a malignant tumor andsimultaneously to treat the tumor by the disclosed method ofthermotherapy. In malignant tumors, the gold nanoparticles coated withanti-tumor antibody are absorbed by the tumor and remain in the tissue.In benign tumors, the gold nanoparticles coated with anti-tumor antibodymay be initially absorbed by the tumor, but will not remain in thetissue and after 1-2 days are eliminated by systemic absorption.

In one embodiment, a method of delivering antibody-coated nanoparticlesto a particular locus, or to a pathological site is disclosed. Anexample of a particular locus is the nervous system to treat aneurological pathology, e.g., a neurodegenerative disease. An example ofa particular pathological site is a known tumor site, e.g., a breasttumor. Administration is either directly to or at the site (i.e., directinjection at the site) or indirectly (e.g., by injection into thebloodstream or other fluid surrounding a site, e.g., cerebrospinalfluid, ocular fluid, ventricles of the brain, etc.). Any site isamenable to the inventive method due to antibody directed targeting orlocalization. For tumor targeting, the method includes but is notlimited to a superficial tumor site. Thus, the following examples arepossible. The tumor may be located on an internal or external anatomicalsite, e.g., skin, extremities, breast, head, neck, and the like. Thetumor may affect any organ, e.g., liver, kidney, brain, bone, prostate,ovary, tonsil, thymus, spleen, and the like. The tumor may be located ina deep tissue, such as in muscle, subcutaneous fat cells, intestines,tendons, connective tissue, ligaments, intracranial, and the like. Thetumor may be located at a site affecting body fluids, e.g.,cerebrospinal fluid, ocular cavity, oral cavity, sinus or sinus cavity,lymph nodes, lymphatic vessel; or the site may be a bodily ventricle,e.g., heart ventricle, brain ventricle, and the like; or the site may bea bodily cavity, e.g., bone cavity, uterus, kidney, bladder, pelvis, orthe like.

In one embodiment, the antibody-coated nanoparticles are provideddirectly to the circulatory system, i.e., either injected into thearterial or venous blood vessel, or delivered by a catheter. In thisembodiment, the method is used to treat pathological conditions of blood(e.g., erythrocytes, leukocytes, platelets, and/or their progenitorcells), and/or to provide systemic treatment, e.g., metastatic sites.

In one embodiment, the antibody-coated nanoparticles are magnetic,diamagnetic, paramagnetic, and/or ferromagnetic.

In one embodiment, the method of providing antibody-coated nanoparticlesto a site, or for systemic delivery, may be combined withimmunologically based B-cells or T-cells that are optionally geneticallymodified. Such B-cells and T-cells may attack localized and/orunlocalized cancer cells or hematological cancers (e.g., leukemias,lymphomas, etc.). In one embodiment, extra-corporal dialysis and/orplasmapheresis may be used in combination with the inventive method ofdelivering antibody-coated nanoparticles to accomplish removal ofexcessive tumor protein in order to protect vital organs, e.g., kidney,liver, brain. In one embodiment, one or more of these therapies areemployed at lower doses when used in combination with the method ofdelivering antibody-coated nanoparticles. In one embodiment, the methodof delivering antibody-coated nanoparticles may have a synergisticeffect with another therapy, e.g., measured by metrics known to a personof ordinary skill in the art.

In other embodiments, nanoparticles of a material other than gold may beused. These include, without limitation, diamond nanoparticles, platinumnanoparticles, combinations of gold, platinum, carbon, and/or diamondnanoparticles. Any of the above nanoparticles may contain at least onehydroxyl group. All such nanoparticles provide the various diagnosticand therapeutic applications as described above for gold nanoparticles.The sizes and shapes are the same as those described for goldnanoparticles. All such nanoparticles may be covalently attached to(poly)ethyleneglycol, i.e., may be PEGylated.

All such nanoparticles create photoacoustic waves when exposed to anexternal energy source such as light, ultrasound, lasers, radiation,microwave, etc. The temperature of the nanoparticles rises and theirmolecules expand. Molecular expansion produces an acoustic sound thatcan be recorded as a photoacoustic wave signal from an in vivo or invitro environment. These sounds, photoacoustic signals, are received byacoustic wave detectors or sensors, and are recorded and analyzed. Suchmethods are known in the art and are similar to signals obtained byendogenous chromophores such as hemoglobin. However, because the goldnanoparticles have stronger absorption of the radiation than otherchromophores, e.g., 30% to 99% higher, less energy is required togenerate an appropriate signal. The signals of each chromophore can alsobe differentiated from other signals by ultrasonic spectroscopy.Acoustic wave sensors have acoustic wave resonator elements includingpiezoelectric material (elements), as known by one skilled in the art.This ability to create and record the acoustic signal is useful fortreating tumors, monitoring treatment efficacy, and making diagnoses,distinguishing malignant cells from benign cells, etc. in the same wayas previously described for optical tissue. For example, the absence ofmalignant cells does not generate an image in photoacoustic imaging, andgenerates a different acoustic signature or characteristic in ultrasoundspectroscopy. Thus, the method can evaluate the presence or absence ofmalignant cells in a human patient. An antitumor antibody-labelednanoparticle, and a temperature indicating substance, in administered tothe patient to form an antibody-labeled-nanoparticle-cell complex at thetumor site. Then, the tumor site is exposed to a radiation energy sourceunder conditions sufficient to achieve a temperature of the complexbetween 41° C. to 56° C. The acoustic sound produced from thenanoparticle at the site is evaluated and correlated with the presenceor absence of malignant cells at the target site by photoacousticimaging and/or ultrasound spectroscopy.

One embodiment using the above-described labeled nanoparticles is amethod to differentiate a tumor or lesion containing malignant cellsfrom one containing benign cells. The nanoparticles are coated with atargeting agent, e.g., antibodies other identifiers known to one skilledin the art, such that they target specific cells when intravenouslyinjected or otherwise administered into a patient.

Cells that are labeled or tagged with gold nanoparticles show 30-40%increase in the resultant acoustic signal, compared to the untaggedcells if the cells possess pigmentation. However, a sample of blood orother tissue, or a tumor (e.g., from biopsy) may be obtained and thenbleached in vitro to rid the cell of pigment, e.g. melanoma cells. Thesamples are exposed to an external energy source (light, laser, etc) andthe resulting photoacoustic wave signals are measured and analyzed usingan ultrasonic spectrometer or other comparable device to determine thepresence or absence of the malignant cells. The bleaching step is notneeded for the majority of the neoplasms to generate photoacousticsignals, e.g., between a normal cell versus one tagged with goldnanoparticles. to be significant. The differences in the opticalabsorption of, e.g., light energy, permits differentiation, in vivo andin vitro, between normal and metastatic lesions (tumor cells).

In vitro acoustic cell analysis is performed by taking a biologicalsample from a patient, e.g., a known volume of blood or other body fluidthat may contain circulating malignant cells, e.g., cerebrospinal fluid(CNS), lymphatic fluid, etc. To this fluid sample is added a specificantibody-coated gold nanoparticle. Because of the specificity of theantibody to a particular cellular receptor, protein, or other bindingtarget, the gold nanoparticle binds to the specific cells present in thefluid and is detected, quantitated, monitored, etc. In one embodiment,an optional temperature-indicating substance is added for in vitroevaluation of the circulating tumor cells in the absence of nanoparticleinjection. This is useful to indicate is metastatic cells are beingreleased, because a positive result can initiate a search for potentialmetastatic lesions even if they have not progressed to a size so as tobe visible by standard means of examination such as computed tomography(CT) or magnetic resonance imaging (MRI). The sensitivity threshold ofrecognizing a metastatic lesion is greater than about 1 mm diametertumor, compared to a sensitivity threshold of greater than about 10 mmdiameter tumor with standard means of examination. In either embodiment,the fluid is then analyzed by, e.g., an ultrasonic spectrophotometer oranother appropriate device, to measure the number of tagged cells in thefluid. This procedure and analysis can also be adapted for use for othertypes of body sample such as tissue biopsies as known to one skilled inthe art.

In one embodiment, a patient-specific antibody to a tumor is prepared.After biopsy, a specific antibody to the malignant cells of the tumor isgenerated. The antibody is then coated with gold nanoparticles resultingin an antitumor antibody-labeled nanoparticle. In one embodiment, abiphasic antibody that binds a target via its Fab region is prepared. inone embodiment, an antibody that binds a target via its Fc region isprepared. Such procedures are known to one skilled in the art. Theseantibody-coated nanoparticles are specific for a specific tumor cellpresent in the patient forming an antitumor antibody-labelednanoparticle-cell complex. In one embodiment, the complex may contain acompound, e.g., an antivascular endothelial growth factor and/or anantiproliferative agent. In one embodiment, the antibodies may also belabeled with a fluorescent dye.

As known to one skilled in the art, tumor cell membranes containnumerous receptors. In one embodiment, multiple antibodies againstindividual antigens may be generated from a tissue biopsy sample.Coating various gold nanoparticles with specific antibody against thetumor cell permits treating a single tumor cells with multiple goldnanoparticles, either alone or with conventional chemotherapy and/orradiation therapy, thus enhancing the tumor-destroying potential of themethod. In one embodiment, the combination of targeted hyperthermia plusradiation, etc. provides conditions in which lower than typical levelsof radiation are needed to destroy tumors, reducing radiationside-effects.

The above in vitro method and analysis can also be used to determine thetemperature (energy) needed to kill tagged malignant cells, or othercells of interest, without damaging the surrounding normal cells. Thisparameter is incorporated in an in vivo procedure to treat specifictumor or circulating malignant cells. For example, a tumor cell lackingpigmentation (e.g., breast cancer cells) may be killed using lowerenergy levels than a tumor cell having pigmentation (e.g., melanomacells).

In one embodiment, a collar positionable to fit on or around a patient'sneck or extremities, contains a multiple diode laser that emits specificwavelengths of radiation energy to heat the labeled nanoparticles to atemperature sufficient to thermally destroy the tumor cells and theycirculate in the area encompassed by the collar. Other devices that canbe used outside or implanted in the body as known to one skilled in theart may incorporate the method for treating and/or destroying malignantcells within other compromised areas of the body such as those havingtumors. Because the temperature generated inside the cells tagged withgold particles exceeds that of exposed non-tagged normal cells, thismethod protects normal cells while damaging the malignant cells.

In one embodiment, the energy source is a free electron laser to providethe hyperthermal treatment. As described, the emitted energy from thefree electron laser heats the nanoparticle of the described compositionsto the desired temperature.

In one embodiment, the method uses magnetic energy to provide thehyperthermal treatment. This embodiment is used for applyinghyperthermal treatment to a lesion that is not accessible to applicationof light energy. Examples of such inaccessible lesions include braincells, or cells deep inside the body.

In this embodiment, magnetic nanoparticles are used. In one embodiment,ferromagnetic, e.g., iron oxide or quantum dot, nanoparticles are used,either alone or combined with other magnetic materials or paramagneticmaterials such as tungsten, cesium, aluminum, lithium, manganese,sodium, platinum, and/or oxygen. Such magnetic nanoparticles areprovided to a patient and exposed to an energy source that provides amagnetic field (e.g., coil, wire) that reverses the direction of thefield rapidly using alternating current. In one embodiment, theparticles are injected locally, e.g., inside a tumor. In one embodiment,the particles are administered intravenously. In one embodiment, theparticles are both injected locally and administered intravenously. Inone embodiment, the described composition is introduced via anaccessible cavity, such as oral, respiratory, or genitourinary, byinjection or catheter. The particles heat up and expand, producingdetectable acoustic (sound) waves. In one embodiment, the magnetic fieldranges from about 0.0001 Tesla (T) to about 13 T. In one embodiment, themagnetic field ranges from about 0.0001 Tesla (T) to about 11 T. Thedegree of heat produced upon such exposure induce an expansion of thenanoparticles that is recorded by an ultrasonic transducer, aspreviously described.

In one embodiment, once delivered to the tumor site, or systemicallyinjected or delivered, the particles are treated with an alternatingmagnetic field and/or electromagnetic radiation to heat or raise thetemperature at the site. In one embodiment, the alternating magneticfield is from 0.0001 T to 13 T, or from 0.0001 T to 11 T, and thealternation frequency is from about 1 GHz to a few seconds, e.g., about10 seconds, about 5 seconds, or about 2 seconds. In one embodiment, atemperature ranging from about 39° C. to 58° C. is achieved. In oneembodiment, a temperature ranging from about 39° C. to about 45° C. isachieved. In one embodiment, a temperature ranging from about 42° C. toabout 47° C. is achieved. In one embodiment, a temperature ranging fromabout 47° C. to about 49° C. is achieved. In one embodiment, atemperature from about 50° C. to about 58° C. is achieved. In oneembodiment, the treatment is effected such that the increasedtemperature is maintained for a period of time ranging from about 5seconds to 30 minutes. In one embodiment, the treatment is effected suchthat the increased temperature is maintained for a period of timeranging from about 1 minute to about 30 minutes. In one embodiment,longer times of maintenance at the increased temperature are used.

In one embodiment, simultaneous or substantially simultaneous imaging ofthe site, e.g., tumor, is effected by means known to the person ofordinary skill in the art, e.g., photoacoustic imaging, magneticresonance imaging, X-ray imaging, optical coherence tomography,ultrasound imaging, fluorescence imaging, positron imaging, surfaceenhanced Raman spectroscopy (SERS), and the like. In one embodiment, themethod of delivering antibody coated nanoparticles to a site isoptionally combined with other therapeutic or medical techniques, e.g. achemotherapy, radiation therapy, anti-vascular endothelial growth-factortherapy, steroid therapy, and the like.

The magnetic particles are attached to an antibody, as previouslydescribed, forming ferromagnetic-antitumor antibody-labelednanoparticles. These particles provide diagnostic and/or therapeuticthermal heating at a biological target or lesion. A temperatureindicating substance is also labeled with the antibody of interest, theferromagnetic particle, or a combination of both.

As one example, a solution or formulation containing theferromagnetic-antibody labeled nanoparticles is administered byinjection to a human or animal patient or by topical application. Theantibody, upon binding to its cellular target, forms aferromagnetic-antibody labeled nanoparticle-cell complex. After a periodof time to allow clearance of the unbound ferromagnetic particles fromthe patient, the patient is placed in a magnetic field. In oneembodiment, this clearance time is less than 2 hours. In one embodiment,this clearance time is 2 hours. In one embodiment, this clearance timeranges between 24 hours to 48 hours. In one embodiment, this clearancetime is up to 48 hours. In one embodiment, this clearance time isgreater than 48 hours.

Clearance can be assessed the determining the presence of circulatingnanoparticles in the patient as follows. A small volume of patient'sblood is placed inside a small electric coil (0.0001 T-0.1 T) in vitro.Altering the magnetic field heats the particles and generatesphotoacoustic images measured by the ultrasonic transducer, whichindicates the present of circulating nanoparticles. The pharmacokineticof these circulating particles can be measured. When the level ofcirculating nanoparticles are significantly reduced as describedpreviously, e.g., 2 hours-24 hours post injection, the patient ispositioned inside an a magnetic resonance imaging (MRI) instrument orany other applicable instrument known to one skilled in the art.

The applied magnetic field heats the nanoparticles, including thesessile or attached particles to the tumor. The procedure isautomatically controlled by photoacoustic imaging monitoring, assubsequently described below. The degree of heat production in theparticles induces an expansion of the nanoparticles that is recorded byan ultrasonic transducer previously described. The patient is maintainedin the magnetic field until a diagnostic or therapeutic temperature of42° C. to 56° C. is reached. The time to reach this temperature dependsupon the energy applied in one session. The session can be repeated asdesired. Generally, the exposure time may vary from <1 minute to >10minutes. The acoustic sound produced from the nanoparticle at the siteis evaluated and correlated with the presence or absence of malignantcells at the target site by photoacoustic imaging and/or ultrasoundspectroscopy. A photoacoustic image is created by exposing the tissue orsite to electromagnetic radiation, e.g. radiation from 190 nm to about10 μm; or microwave and radiofrequency radiation produced, e.g., using alaser. If the material has magnetic properties, by reversal of themagnetic field, one can heat the tissue or site; one can also usemechanical waves produced by another ultrasound transducer to heat thetissue or site. These mechanisms create thermoelastic expansion of amolecule that generates a wideband (MHz) emission. The time andamplitude of the photoacoustic images provide information regarding theabsorption and location of the source, depending on the degree ofthermal expansion of the material, etc., as known to one skilled in theart.

In one embodiment, the method uses magnetic energy to provide thehyperthermal treatment. This embodiment is used for applyinghyperthermal treatment to a lesion that is not accessible to applicationof light energy. Examples of such inaccessible lesions include braincells, or cells deep inside the body.

In this embodiment, magnetic nanoparticles are used. In one embodiment,ferromagnetic, e.g., iron oxide, nanoparticles are used, either alone orcombined with other magnetic or paramagnetic materials. In variousembodiments, the described nanoparticle, e.g., ferromagneticnanoparticle, is carbon coated by methods known in the art, making thenanoparticle more biocompatible, e.g., less toxic. Such magneticnanoparticles are provided to a patient and exposed to an energy sourcethat provides a magnetic field (e.g., coil, wire) that reverses thedirection of the field rapidly using alternating current.

The following methods control the energy applied to the nanoparticles,indicating when to treat after injection of the nanoparticles. Anoncoagulative laser or radiofrequency or an acoustic wave, whenabsorbed, can produce a photoacoustic ultrasonic emission of about 1 MHzor more (two- or three-dimensional), creating an image of a tissue,site, etc. The image, in the form of a graph, curve, etc., shows anamplitude that depends on the amount of thermal energy absorbed orproduced at the tissue or site. One can record this temperature increaseas an image, regardless of the exact degree increase. Thischaracteristic can be used to distinguish low and high energy applied,e.g. internally generated, by magnetic resonance on the ferromagneticparticles, and can be used to modify the amount of energy required. Acomputer processor (digital signal processor) can transmit the desiredsignal from the ultrasound unit to a device, e.g., a laser,radiofrequency device, magnet, etc., as known to one skilled in the art,to increase or decrease the applied energy to achieve potentially anydesired temperature for any given situation and site. This energyvaries; relatively low heat up to 55° C. for non-coagulative lasertreatment, to >60° C. for coagulative laser treatment, to >100° C. inone embodiment and >500° C. in one embodiment for photoablative lasertreatment, i.e., very short pulses but very high energy, depending onhow deep the target tissue is located inside the body, and the propertyof the energy absorber inside that targeted tissue. This method resultsin a treatment that is visible, adjustable, and automatic depending onthe needed temperature regardless of the depth of the lesion inside thebody. Previously only a light source with an external photographicdevice could indicate the surface temperature. However these devices andwavelengths are limited by their wavelength, tissue penetration, andtissue water content, limiting light transmission to a few millimeters.

Photoacoustic spectroscopy is very sensitive if a tissue contains anygas. Because animal tissue always contains gases as oxygen, nitrogen,and/or carbon dioxide, any degree of temperature increase causesexpansion of these gases at that location. The sensitivity ofphotoacoustic spectroscopy can reach one part in trillion levels.Therefore this method demonstrates the thermal effect that was generatedon the specific tissue, and the energy-absorbing nanoparticles can beimaged inside the body using this method.

As one example, a solution or formulation containing theferromagnetic-antibody labeled nanoparticles is administered byinjection to a human or animal patient human patient. The antibody, uponbinding to its cellular target, forms a ferromagnetic-antibody labelednanoparticle-cell complex. After a period of time, such as at least 2hours up to 48 hours or more to allow clearance of the unboundferromagnetic particles from the patient, the patient is placed in amagnetic field, such as that provided by an magnetic resonance imaginginstrument or any other applicable instrument known to one skilled inthe art. In various embodiments, the magnetic field is applied to aportion of the body, e.g., locally at the target site, or to the wholebody.

The applied magnetic field heats the nanoparticles. The degree of heatproduction in the particles induces an expansion of the nanoparticlesthat is recorded by an ultrasonic transducer previously described. Thepatient is maintained in the magnetic field until a diagnostic ortherapeutic temperature of 42° C. to 56° C. is reached. The heatproduction is controlled to achieve this temperature and hence to treatonly tumor cells. The acoustic sound produced from the nanoparticle atthe site is evaluated and correlated with the presence or absence ofmalignant cells at the target site by photoacoustic imaging and/orultrasound spectroscopy.

In embodiments, the described photoacoustic imaging may be combined withother imaging techniques such as X-ray, MRI, CT, and PET scans. Ininstances where the tumor is light accessible, optical coherencetomography (OCT) combined with photoacoustic imaging may be used.

In various embodiments, the method comprises providing a describednanoparticle composition, e.g., a nanoparticle labeled with an antibody,and providing an energy source which causes the nanoparticle to increasein temperature, resulting in heating tissue and cells in the tissue ofan animal, particularly a human patient, at least to the temperaturesufficient to kill or damage the cells, in conjunction withphotoacoustic imaging and/or ultrasound spectroscopy, where a desiredtemperature is achieved using an energy source and an imaging technique.As described, an energy source, such as a magnetic energy source, can beused which substantially penetrates the tissue, and thus allowsactivation of nanoparticle compositions, e.g., ferromagneticnanoparticles, which may be located within deep tissue sites, or anenergy source, such as light, may be used for surface or sub-surfaceactivation of a nanoparticle composition. For example, in cases ofsurface tumors or tumors accessible by catheter, a carbon nanoparticlecomposition may be used which is activated or heated using light orelectromagnetic radiation.

In one embodiment, any of the described methods for hyperthermallytreating cells, including the use of magnetic or gold nanoparticles, maybe combined with platelet-derived therapy. In one embodiment,platelet-derived therapy comprises obtaining platelets from thepatient's blood, coating the platelets with antibodies, andreintroducing the coated platelets into the patient. In one embodiment,the antibodies are anti-tumor antibodies. In one embodiment, the coatedplatelets are reintroduced by injection into the patient's circulatorysystem after an initial hyperthermia therapy. The coated plateletstravel though the blood vessels reaching the tumor surface where, due tothe prior hyperthermia-induced tumor cell damage, the injected coatedplatelets attach to the tumor and its associated vascular supply,creating a blood clot or thrombus, which obstructs the blood supply tothe tumor, and nutritionally starves the tumor. In one embodiment,additional or subsequent hyperthermia therapy treatments may beconducted during or following the platelet-derived therapy. In oneembodiment, the platelets are coated with an antibody. This may beaccomplished by methods as disclosed in, e.g., Iyer et al. (Meth. Mol.Biol. 2011; 751:553-563, Single-step conjugation of antibodies toquantum dots for labeling cell surface receptors in mammalian cells,which is incorporated by reference in its entirety herein. Using suchmethods, cell surface receptors in platelets are labeled usingantibody-conjugated semiconductor quantum dots. In one embodiment, thequantum dots are ferromagnetic, and as described above, upon exposure toa magnetic energy source, provide hyperthermia therapy. The hyperthermiatherapy provided by the platelet-coated antibody-quantum dot conjugatecan be in addition to the hyperthermia therapy provided by thenanoparticle, or it can be the sole source for the hyperthermia therapy.In one embodiment, any of the described methods and compositions, suchas hyperthermia treatment with photoacoustic imaging, may be used fortreating diseases or infections due to bacterial, fungal, viral, and/orprotozoan organisms. In one embodiment, a described method may besystemically applied, e.g., intravenous administration of the describedcomposition. In one embodiment, the nanoparticle is labeled with anantibody which recognizes the infectious agent. In one embodiment, thenanoparticle is a ferromagnetic nanoparticle. In one embodiment, any ofthe described nanoparticles are conjugated with an antibody that bindsto, or associates with, an infectious agent, e.g., an anti-bacterialantibody, anti-viral antibody, anti-fungal antibody, anti-parasiticantibody, and the like. In this embodiment, the conjugated nanoparticlecomposition is locally or systemically administered and subjected toradiation, such as electromagnetic radiation or alternating magneticfield, to achieve temperatures of between about 40° C. to about 49° C.in one embodiment, or between about 42° C. to about 49° C., or higher asneeded to destroy the micro-organism(s). The toxins from the infectiousagent can be removed from the blood through a dialysis system. In oneembodiment, the method may be combined with additional therapies,including antibiotics, anti-fungal agents, anti-viral agents, etc. Invarious embodiments, following the method, the patient's blood isprocessed through an extracorporeal dialysis system, which removes orreduces the infectious agent, e.g., bacteria, and their associatedtoxins from the blood. In one embodiment, the dialysis system furthercomprises a magnetic separator, which allows separation and collectionof nanoparticles, e.g., ferromagnetic nanoparticles, along with damagedinfectious agent, e.g., bacteria, from the blood. In embodiments where atumor is subjected to the described hyperthermia treatment, thecirculating tumor cells can be collected by the magnetic separator alongwith the nanoparticles in the dialysis machine. In another embodiment,the patient's blood is exposed extracorporally to a localizedalternating magnetic field prior to the blood being processed by adialysis machine, such that the infectious agent, their associatedtoxins, and/or damaged tumor cells are separated and removed beforerecirculating the blood into the patient again, which prevents releaseof toxins inside the patient's body.

In one embodiment, any of the described methods and compositions, suchas hyperthermal treatment with photoacoustic imaging, are used toameliorate a neurodegenerative disease, e.g., Alzheimer's disease,Parkinson's disease, and the like. In one embodiment, the describednanoparticle, such as colloidal gold or ferromagnetic, is conjugatedwith an antibody that binds to, or associates with, beta amyloid peptidewhich forms plaques found in the brain of Alzheimer's patients. In oneembodiment, the neurodegenerative disease-targeted nanoparticle isinjected into the cerebrospinal fluid. The neurodegenerativedisease-targeted nanoparticle may be subjected to electromagneticradiation, such as microwave or RF, or alternating magnetic field, toachieve temperatures ranging between about 45° C. to about 55° C., for aperiod sufficient to reduce or prevent aggregation of beta amyloidpeptides and/or to destroy beta amyloid peptide aggregates.

In one embodiment, the nanoparticle is colloidal gold. In variousembodiments, the shape of the colloidal gold nanoparticle may bespherical, rod-shaped, irregular, etc. In one embodiment, the goldnanoparticle is rod-shaped, e.g., a nanorod, and may be subjected tosurface plasmon resonance (SPR), which may be used as an imagingtechnique and/or as a means for heat generation for hyperthermiatreatment, depending on the wavelength of the electromagnetic energy,e.g., near infrared light, and the axial diameter of the nanorod.

In one embodiment, the nanoparticle used in any of the described methodshas (poly)ethylene glycol (PEG) groups attached. Methods to conjugatewith PEG are known in the art and include, but are not limited to,encapsulating the nanoparticle and/or associating PEG with thenanoparticle. In embodiments, a targeting group is associated with theconjugated PEG; examples of such targeting groups include but are notlimited to an antibody or a portion thereof, a ligand for a receptor, orother moieties that promotes association, retention, targeting,transport, and/or uptake of the described nanoparticle.

In one embodiment, the described nanoparticle, e.g., a pegylatedcolloidal gold or ferromagnetic nanoparticle, is configured as a drugcarrier. In this embodiment, the nanoparticle is associated with a drugand facilitates or effects drug delivery by, e.g., drug administration,dispersion, targeting, controlled delivery (e.g., time-release), and thelike. For example, hydrophobic drugs often require encapsulation orother means in order to increase the drug water solubility. In oneembodiment, a drug is associated with a nanoparticle, such that theassociation may be covalent or non-covalent; in the case of a covalentassociation, the drug may be tethered to the nanoparticle by a linkingmoiety. In one embodiment, a drug-associated nanoparticle is used in oneof the described methods, such as hyperthermal treatment, in combinationwith a role as a drug delivery vector, such that, e.g., a target site(e.g., tumor) may be subjected to both hyperthermal treatment and drugtreatment,

In one embodiment, a thermoacoustic imaging technology is used with thetumor cells and tumor stem cells. Known photoacoustic technology useslight-induced heat, producing expansion, to create an ultrasonic soundthat is recorded by an ultrasonic transducer. The site is imaged aspreviously described. Photoacoustic technology is, however, limited bythe penetration of the electromagnetic wavelengths from ultraviolet (UV)to far infrared (IR). The maximum penetration depth depends on thewavelength of the light that penetrates the tissue, e.g., 1-2 cm. Evenwith photoacoustic tomography (PAT), the maximum depth that can beimaged in the tissue is about 7-8 cm.

With the use of thermoacoustic imaging using heat sensitiveantibody-coated nanoparticles and heating the coated-nanoparticlecomplex (e.g., microwaves, radiofrequency waves, focused ultrasound,etc.) one can image the thermal expansion of intended tissue, e.g.,tumor, etc. regardless of the tumor depth in the body. The same conceptapplied for creating an image using “photoacoustic”/thermoacousticimaging, using magnetic antibody-coated nanoparticles with a reversiblemagnetic field. In both of these embodiments, MRI thermal imaging isalso able to image the thermally treated tissue. Similarly one can heatthe desired tissue with a focused ultrasound with one transducer, andobserve the temperature rise through the photoacoustic technology.

As described above, in various embodiments, a combination ofhyperthermal treatment and imaging is provided. In one embodiment, thenanoparticle is colloidal gold which allows for hyperthermal treatment,as described above, as well as imaging using SERS. In variousembodiments, the colloidal gold nanoparticle is associated with quantumdots and/or Raman reporters. In one embodiment, the colloidal goldnanoparticle, associated with quantum dots and/or Raman reporters, isalso associated with or encapsulated by PEG. Further, as describedabove, the PEG may be further associated with a targeting moiety, suchas an antibody or a portion thereof.

Thermotherapy Using a Magnetic Conformer

Magnets are found in nature and can be formed into any shape. Allmagnets have the characteristics of absorbing other magnetic elements,e.g., iron. Electromagnets are produced by wrapping a wire as a coilaround iron of any shape, size, or conformation. In an electromagnet themagnetic pole depends on the direction of the electrical flow in thesurrounding wire and remains stable as long as electricity flows in thesame direction. If the electrical flow is turned off, the electromagnetloses its magnetism. If the direction of the current is changed, themagnet changes its polarity and becomes an alternating magnet.

In one embodiment, the method uses a magnetic conformer. This conformercan be a natural magnet or an electromagnet in which the magnetic fieldis not reversed. In such an embodiment, the treatment modality isapplied either locally, systemically, or both locally and systemicallydepending on the lesion and malignancy. When the treatment modality isapplied systemically, the tumor has potentially metastasized in the bodyand the entire body is in need of thermotherapy. The localized magnetwith stable magnetic field collects the circulating ferromagneticnanoparticles or nanowires in the tissue near its magnetic field.Nanowires, so-named because the width is smaller than the length, can bemetallic (e.g., Ni, Au), semiconductor (Si, InP, GaN, etc.), organic, orinorganic and have greater efficiency converting light to energy.

In one embodiment, the localized magnet with a stable magnetic fieldattracts the ferromagnetic radioactive and/or drug coated nanoparticlesor nanowires in tissue or in a benign or malignant tumor near itsmagnetic field. As in the general method disclosed herein, thenanoparticles or nanowires may be coated with anti-tumor antibody,contain therapeutic agent(s) (e.g., genetic agent (DNA, RNA),radioactive agent, chemical agent (chemotherapeutic, antimicrobial,etc.)) in any combination.

In one embodiment, the photoacoustic or thermoacoustic component of thethermotherapy system described herein is connected to a processor thatis connected to the thermal energy producing system (FIG. 6). Thisembodiment permits specific control of the amount of energy deliveredand maintained at the lesion site to create a specific temperature atthe nanoparticle- or nanowire-cell complex site.

In one embodiment, the treatment modality is applied either locally orsystemically but preferentially localized to a defined or partiallydefined tumor site with no systemic metastasis. The method is furtherdisclosed using a choroidal melanoma of the eye, but as one skilled inthe art will appreciate the method is not so limited and is applicableto practically any part and/or body system of the body (skin, mucosa,prostate, brain, vertebra, extremities, urogenital system, endocrinesystem, gastrointestinal system, pancreas, breast, etc.).

The general use of radioactive nanoparticles in a method for treatmentof eye tumors has been fully disclosed previously. In the embodimentcurrently described, magnetic nanoparticles or nanowires and aconforming magnet, also termed a magnetic conformer, are used inconjunction with photoacoustically controlled temperature measurement.This is achieved by connecting the electromagnetic conformer, in thiscase as the source of energy delivery, to a processor that is in turnconnected to the photoacoustic system, to adjust the desired temperaturelevel at the lesion site and is to a predetermined level. As previouslydisclosed, energy sources can be electromagnetic radiation, microwaveradiation, radiofrequency (RF) radiation, ultrasound radiation, analternating magnet, etc.

In one embodiment using an electromagnet, a simple magnet is positionedexternally at or near a tumor site, or internally at or near a tumorsite by a minimally invasive surgical procedure to attract circulatingferromagnetic nanoparticles or nanowires. The magnet size depends on thetumor or lesion size. For positioning in the eye, the magnet will berelatively small in comparison for positioning in or near breast, bone,or soft tissues. The magnet shape, like magnet size, is variable andflat, concave, semicircular, circular, annular etc. shapes may be useddepending on the tumor or lesion location.

The simple magnet is an electric coil that is positioned around a metal,with a positive pole and a negative pole. To produce an alternatingmagnet, an electrical switch is used to rapidly change the direction ofelectricity and change the magnetic pole of the metal with analternating frequency of 1 per second to 1 per nanosecond and producinga magnetic field of, e.g., about 1×10⁻⁶ Tesla to 10 Tesla. Prior toconverting the magnet to an alternating magnet, the nanoparticlescirculating in the blood or lymphatic fluid or provided at a specificsite are concentrated in the area of the localized magnet with a stablemagnetic field.

Using the electrical switch, the magnet is made to rapidly alternate itspolarity, i.e., producing an alternating magnetic field. Thenanoparticles or nanowires located in the alternating magnetic field ofthe coil develop a semicircular motion. The rapid polarizationalternation creates heat in the nanoparticle or nanowire. Because thenanoparticles or nanowires are concentrated at or near the tumor site,the heat that is generated heats the nanoparticle-cell or nanowire-cellcomplex at or near the target site.

In one embodiment, the nanoparticles are injected into the circulatorysystem. While other administration methods are within the inventivescope, the ease and convenience of direct intravenous injection, or lessroutinely intraarterial, injection makes this route attractive andprovides a rapid route to reach all organs. Topical, intrathecal,subcutaneous, submucosal, inhalation, oral, cerebrospinal fluidinjection, or intracavity (bladder, uterus, etc.) administration routesare possible to directly reach tumors or lymph nodes.

Regardless of the route of administration e.g. intravenously, of thenanoparticles, once in the body they are attracted selectively more to amagnet that is positioned in the localized area. Using the eye as anon-limiting example only, the conforming magnet can be placedexternally on, or provided on a small probe to, an accessible ocularlesion, or the conforming magnet may be implanted on an internal lesion,e.g., under the conjunctiva, over the sclera, etc.

After a period of time post administration of 1 min to 15 min,sufficient to concentrate the nanoparticles or nanowires at a targetsite, they are preferentially treated by the disclosed method ofthermotherapy. The presence of the magnetic conformer converts theinitially stable magnet to a reversible magnetic capable of generatinglocalized heat in the nanoparticle-cell or nanowire-cell complex,without displacing their location. In alternative embodiments, or afterremoving the localized magnet, the lesion or whole body undergoeshyperthermal treatment generated by means other than a conformingmagnet, e.g., electromagnetic radiation, microwave radiation,radiofrequency radiation, etc., with the controlled temperature usingthe photoacoustic processor system as previously described herein indetail.

In either embodiment, hyperthermal treatment involves generating heatfrom 37° C. degree to <60° C. in stepwise 1° C. to 3° C. incrementsgradually as desired for release of agents from the nanoparticle anddamage to the tumor cells. The polymers carrying the agents release themduring the heating process, i.e., when the temperature for the polymerstretching or melting occurs sufficient to release the agent. Inembodiments where a combination of treatment modalities are applied, thecombination of agent therapy and radiotherapy with thermal therapyprovides synergistic treatment for the lesion or tumor.

The duration of therapy can vary from 0.5 minute to 1-2 hours, dependingupon the duration and therapy needed, as well as patient parameters, asone skilled in the art will be able to determine. In tumors that havemetastasized, thermal treatment may be applied to the whole body afterlocalized therapy. Using again the illustrative and non-limiting exampleof use of the method to treat an ocular lesion, the conformer can bedirectly applied on the cornea or the conjunctiva when a lesion isexternally located. To treat ocular lesions that are not externallylocated, e.g., tumors located in the back of the eye or close to theoptic nerve, a conjunctival incision is used to provide the conformingmagnet to the area. The incision size depends upon the lesion size, andthe magnet can be precisely located on or near the lesion site using,e.g., ophthalmoscopy, ultrasound, MRI, CT scan, PET scan, etc., as knownto one skilled in the art. Once the location is verified, the magneticconformer is temporarily placed and stabilized, e.g., sutured in place,to the scleral surface over or in the vicinity of the lesion for adesired time sufficient to accumulate an adequate amount ofnanoparticles or nanowires at that site. The treatment can be repeatedat any time as desired, and gauged by use of one or several imagingmethods described.

Exsosomes are univesicular or multivesicular structures originating fromthe plasma membrane of all cells, including cancer cells. Exosomes servein cellular waste management, but can also function between adjacentcells in intracellular signaling, and as a conduit to transfer geneticmaterial between cells. In some circumstances, exosomes can “infect”adjacent cells or travel in the circulation and facilitate a metastaticprocess. Booth et al., J. Cell Biol. 172 (2006) 923.

Exosomes are much smaller than the cells from which they originate,typically exosomes are between 30 nm-100 nm. Their membrane, similar tothose of other cells, is composed of protein and lipid layers. Exosomescarry proteins and genetic components (e.g., RNA, DNA) and have the samesurface receptors as the cells from which they originate.

Exosomes thus carry biomarkers of their cell of origin, either a normalcell or a cancer cell, and thus can serve as a marker for the existenceof cancerous cells. However, exosomes also may be used to evaluate theprognosis of a pathology, e.g., malignant tumors, infectious organisms,viral infection diseases, etc. An increase of exosomes originating fromcancerous cells in the vascular system can be an indicator that a canceris more of an aggressive type. The presence of exosomes in thevasculature may facilitate metastasis. There is no known therapy totarget such exosomes, either circulating or localized.

An embodiment of the invention is a method using hyperthermal therapyapplied to target either circulating or localized exosomes. Thisembodiment of the method simultaneously recognizes, quantifies, anddestroys or reduces the number of exosomes, using hyperthermal therapy.The method is useful for both diagnostic and therapeutic applications.Quantification of exosomes is used for diagnosis of, e.g., a malignancy,an infection, etc., and can be performed either in vitro and/or in vivo.Destruction or reduction of exosomes is accompanied by destruction orreduction of the cancer cells from which they are produced, and is usedin therapeutic applications.

In cancer immune therapy, the patient's circulating tumor exosomes canbe cultivated, using tissue culture methods known in the art, with apopulation of the patient's growing defensive cells: antigen-presentingcells (APC), dendritic cells, and/or macrophages in order to sensitizethese defensive cells. Upon intravenous injection back into the patient,these now sensitized defensive cells activate the patient's own T cellsto attack both the tumor cells and the circulating exosomes originatingfrom the tumor cells. The disturbed microenvironment of the tumor afterthermotherapy using labeled nanoparticles and/or quantum dots contributeto making the remaining tumor cells accessible to the sensitized Tcells. In addition, RNA and DNA is released from the damaged exosomes inthe serum and, in turn, is damaged by oxidative molecules such ashydroxyl radicals and other reactive oxygen species in the serum,important for treatment of viral and infectious diseases.

The nanoparticles and/or quantum dots may contain polymers such as(poly)ethylene glycol, i.e., they may be PEGylated, to increase theirlongevity and biocompatibility, as subsequently described. Thenanoparticles and/or quantum dots may be associated with cellpenetrating peptides to enhance their uptake in the tumor cells. As onlyone non-limiting example, the nanoparticles and/or quantum dots may beassociated and/or coated with a thermosensitive polymer (e.g., chitosan,chitin, etc.) to deliver compounds that are anti-tumor or anti-canceragents. Such compounds include, but are not limited to, antineoplasticagents. Such agents include drugs and/or biologics. In one embodiment,the nanoparticles and/or quantum dots can be conjugated with humanizedantitumor monoclonal antibodies designed for the treatment of cancersand for the treatment of bacterial, viral, and parasitic infections.

The content of exosomes can influence the function of the cells in whichthey come into contact, or in which they are taken up. A plurality oforganic, non-organic, or synthetic nanoparticles alone or, with aplurality of quantum dots, are associated with various compounds toenhance their biocompatibility. Such compounds are known in the art andinclude, but are not limited to, (poly)ethylene glycol (PEG) and variousPEG moieties, organic molecules, proteins such as biotin, shortpeptides, naturally occurring amino acids such as arginine orphenylalanine, mono- or bilayers of phospholipids, or biotargetedmolecules etc. Association includes coatings, covalent attachments, andother types of associations sufficient to enhance biocompatibility. Forexample, the nanoparticles may be PEGylated to increase their longevity.The nanoparticles can consist of both inorganic or organic materials, aspreviously described. The particles can be entirely or partiallybiodegradable. The particles may also be included in or coated on abioabsorbable or non-bioabsorbable but biocompatible polymer.

The nanoparticles are also conjugated with antibodies against the cellmembrane's multiple exosome receptors and tumor cell membrane receptors.The conjugated and biocompatible nanoparticles, termed labelednanoparticles, are attached to and/or taken up by both exosomes and themalignant or normal cells.

The labeled nanoparticles or quantum dots can be magnetic, paramagnetic,or nonmagnetic; gold, quartz, silicon, graphene, zinc oxide, ceramic,plastic, Particles of crystalline silicon may be monocrystalline cells,poly or multicrystalline cells, or ribbon silicon having amulticrystalline structure. The particles may be a nanocrystal ofsynthetic silicon, gallium/arsenide, cadmium/selenium,copper/indium/gallium/selenide, zinc sulfide, indium/gallium/phosphide,gallium arsenide, indium/gallium nitride, and are synthesizedcontrolling crystal conformations and sizes. In one embodiment, thenanoparticle may comprise a nanocrystal, such as cadmium/selenium(Cd/Se), and a metal. For example, a CdSe/Au nanometer-sized compositeparticle may be synthesized through a two-step procedure, where CdSenanorods are formed by the reaction of Cd and Se precursors in a mixtureof trioctylphosphine oxide and an alkylphosphonic acid to formrod-shaped CdSe nanoparticles, and the CdSe rods are treated with amixture of gold chloride, didodecyldimethyl-ammonium bromide, andhexadecylamine to stabilize the nanocrystals and to reduce the goldchloride to elemental gold. Because the two ends of the CdSe rods differcrystallographically, and therefore chemically, control of growthconditions allows growth of Au particles preferentially on one end ofeach rod. In addition to CdSe/Au particles, one skilled in the art willreadily recognize that particles can be constructed from a variety ofother semiconductor/metal and semiconductor/semiconductorhetero-junctions. For example, particles based upon semiconductor/metalhetero-junctions between group II-VI, IV, III-V, IV-VI, referring togroups of the periodic table, metal-oxide, or organic semiconductors anda metal, and in particular those based upon Si/Au, GaAs/Au, InAs/Au, andPbS/Au hetero-junctions, can be used in the same way as those discussedhere and one or more combinations of these can be used.

The size of the nanoparticles is between 1 nm-100 nm. In one preferableembodiment, the size of the nanoparticles is between 1 nm-10 nm. In thisembodiment, the nanoparticle size is about an order of magnitude smallerthat the exosome size.

Exosomes circulate in body fluids and, because of their small size, theycross the blood brain or blood ocular barrier and are taken up by normalcells or tumor cells. The labeled nanoparticles attach to the exosomes.

The labeled nanoparticles or quantum dots, when injected into the body,seek the exosomes and cell membrane receptors on cells such as tumorcells. As a result, the nanoparticle or quantum dot-conjugated exosomesare present in the circulation. The nanoparticles and/or quantum dotsare excreted completely in the urine. As a result, the nanoparticleand/or quantum dot-conjugated exosomes can be detected extra corporallyin a body fluid sample, e.g., blood, extra and intraocular fluid,cerebrospinal fluid, sputum, nasal secretions, urine, sweat, etc.

The complex of exosomes and nanoparticles and/or quantum dots can bedetected and quantitated using various methods.

One quantitation and detection method uses that disclosed in U.S. Pat.No. 8,119,165 for detecting circulating malignant cells. The complex ofnanoparticles and/or quantum dots-exosomes are heated using externalthermal energy, e.g., electromagnetic, ultraviolet, visible, infrared,microwave, radiofrequency radiation, ultrasound, reversible magneticfield, etc. or a combination of these. The increased temperature inthese complexes is measured using photoacoustic technology as describedin U.S. Pat. Nos. 8,121,663 and 8,554,296. The thermal energy ispreferentially absorbed by the nanoparticles and/or quantum dots, thusthe temperature of the nanoparticles and/or quantum dots rises much morerapidly than the temperature of the tissues or cells to which they areattached or taken up. The temperature rise causes physical expansion ofthe nanoparticles and/or quantum dots, producing a sound wave that canbe measured, imaged, and counted by a photoacoustic system. Thetemperature variation can also be measured precisely with thistechnique, demonstrating either an increase or decrease of the soundwave amplitude in photoacoustic imaging or tomography. The acousticsignal is measured and related to the temperature of the nanoparticlecomplex to effect hyperthermal therapy. The method can be combined withflow cytometry for cell quantitation. The quantitation and detectionmethod can be used ex vivo, e.g., on body fluid samples.

Another ex vivo quantitation and detection method is by application ofvarious wavelengths of light to the samples. The complex produces avisible response based on light exposure, e.g., fluorescence, and thevisible response can be quantified by a detector and processed by acomputer.

Other ex vivo quantitation and detection methods include standardmethods that include flow cytometry, microscopy, mass spectroscopy,bioformatic analysis, etc.

In one embodiment, the nanoparticle and/or quantum dot complex maycontain or be associated with various therapeutic compounds using athermosensitive coating, e.g., a thermosensitive polymer as known in theart. Therapeutic compounds include, but are not limited to,anti-infective agents, anti-viral agents, anti-parasitic agents,anti-fungal agents, antiproliferative agents, enzyme inhibitors ormodulators, anti-VEGF, gene suppressants, gene stimulators, etc., asknown in the art. In this embodiment, increasing the temperature of thecomplex by hyperthermal therapy of nanoparticles and/or quantum dots andexosomes to about 39° C.-41° C. releases the compound from the thermosensitive polymers associated with the nanoparticle/quantum dotcarriers. Further temperature increases to 43° C.-45° C. or 47° C.-50°C. or higher adversely affect the exosome membrane, rendering theexosome more vulnerable to the therapeutic agents released from thenanoparticles or administered at lower dose orally or systemically. Thiscontrolled thermotherapy can be combined with other standard therapiesfor cancer, e.g., radiation, immune, or vaccine therapy etc.Simultaneously a second plurality of nanoparticles and/or quantum dotsmay be administered containing other agents, e.g., small molecules,other therapeutics including but not limited to gene therapy, to fightthe disease process.

The temperature increase is related to the amount of energy applied. Thephotoacoustic system measures the temperature of the nanoparticle and/orquantum dot, and communicates by a processor with the energy deliverysystem to control the amount of energy delivery to the complex ofnanoparticles and/or quantum dots and exosomes at a predetermined level.

In one embodiment, the nanoparticles and/or quantum dots providestherapeutic agents in addition to its hyperthermal effect. In thisembodiment, the nanoparticles and/or quantum dots may provideimmune-stimulatory agents, vaccines, and other therapeutic compoundsknown in the art. As only one non-limiting example, the nanoparticlesand/or quantum dots may be associated and/or coated with athermosensitive polymer (e.g., chitosan, chitin, etc.) to delivercompounds that are anti-tumor or anti-cancer agents. Such compoundsinclude, but are not limited to, antineoplastic agents. Such agentsinclude drugs and/or biologics. Antineoplastic drugs include cisplatin,carboplatin, tetraplatin, iproplatin, adriamycin, mitomycin C,actinomycin, ansamitocin and its derivatives, bleomycin, Ara-C,daunomycin, metabolic antagonists such as 5-FU, methotrexate, isobutyl5-fluoro-6-E-furfurylideneamino-xy-1,2,3,4,5,6hexahydro-2,4-dioxopyrimidine-5-carboxylate, melpharan, mitoxantrone,lymphokines, aclacinomycins, chromycins, olivomycins, etc.Antineoplastic biologics include DNA, RNA, protein, siRNA, genes orportions thereof, etc.

In this embodiment the immune response is stimulated, e.g., tofacilitate removal of the hyperthermally-damaged tumor cells and theircirculating exosomes, to target infectious agents, etc.

Exosomes play a role in Alzheimer's disease by facilitating aggregationand accumulation of beta-amyloids proteins. Therapies that facilitatetheir removal would facilitate treatment. Stimulated microglial cellsmay be activated by adding therapeutic agents to the complex ofnanoparticles and/or quantum dots and exosomes. The complex may beadministered orally, systemically, or injected into a body cavity. Theuse of combined nanoparticles and/or quantum dots and exosomes enhancescellular uptake of genes that can provide combined genetic therapy withmore conventional drug therapy, enhancing attach of a disease process.

In one embodiment, neurodegenerative diseases such as Alzheimer'sdisease or Parkinson's disease may be treated by the inventive methodusing nanoparticles and/or quantum dots containing antibodies againstbeta-amyloid protein, e.g. Crenezumab, etc. These nanoparticles and/orquantum dots, which may be 1 nm-5 nm, pass through the blood brainbarrier and penetrate into areas not previously accessible to suchtreatment. This embodiment of the method can be used for treatment ofdegenerative disease of the central nervous system and/or the eye.

In one embodiment, the nanoparticles are coated or otherwise associatedwith an antibody to specific cells, e.g., anti-tumor antibody, and areadministered to a patient in need of therapy. The administerednanoparticles accumulate at a tissue site that is targeted by theantibody through typical antigen-antibody binding. The nanoparticlesaccumulated at the target site are radiated with an energy source,inducing a photoacoustic signal or sound wave from the nanoparticles.Using a processor, the photoacoustic technology controls the amount ofthermal energy delivered at the desired temperature to the target site.The target site tissue is imaged, the temperature is recorded, and thephotoacoustic sound wave is recorded, either from one location externalto the body or from multiple locations. The recorded sound waves areamplified and processed to generate a computational tomographic image ofthe nanoparticles at the tissue target site. Since the antibody-coatednanoparticles are directed to attach to one or more surface receptors ofspecific cells, if the nanoparticles are attached to tumor cells, oneobtains a two- or three-dimensional photoacoustic image of a tumor. Ifthe antibodies are directed to normal cells of an organ, one obtains atwo- or three-dimensional photoacoustic image of that organ regardlessof its location.

In one embodiment, internal organs are imaged using the method by, e.g.,radiofrequency, microwave, alternating magnet, or focused ultrasoundwhich penetrates deep in tissue to controllably heat the nanoparticlesto obtain high-resolution two- or three-dimensional photoacoustic imagesof either a tumor or an organ.

In one embodiment, the temperature from the photoacoustic source iscommunicated to the energy source to control, via the processor, theamount of the energy and its duration as delivered to a desired degreeof temperature measured at the nanoparticle tissue complex. The thermalenergy that is applied to the desired tissue target site, containing theaccumulated nanoparticles, induces a photoacoustic sound. Thephotoacoustic sound is recorded and produced as each of a thermal graphof the target site, and as an image in any of one-, two-, orthree-dimensions.

In one embodiment, a combination of imaging systems may be used.Illustrative and non-limiting examples include photoacoustic temperatureimaging and ultrasound or focused ultrasound, photoacoustic temperatureimaging combined with MRI or functional MRI (fMRI), photoacoustictemperature imaging combined by CT, PET, etc. scan, photoacoustictemperature imaging combined with photography or OCT, photoacoustictemperature imaging combined with alternating magnetic field imaging,etc. Because other imaging systems such as MRI, CT scan, PET scan etc.do not provide a high resolution image, their combination withphotoacoustic imaging results in enhanced image acquisition withimproved resolution.

The thermal energy applied may be in the form of electromagneticradiation, ultraviolet radiation, visible light, infrared light,radiofrequency waves, microwaves, focused ultrasound, alternatingmagnetic field radiation, or a combinations of these. The combined useof various energy delivery systems decreases the amount of energyrequired for each unit or source, which may be desirable. Examples ofcombined therapy systems include, but are not limited to, lasers ofvarious wavelengths, radiofrequency waves, microwaves with focusedultrasound, microwaves with alternating magnets, etc.

In one embodiment, the thermal energy may be applied from multiple sitesto prevent pain to the patient and possible overheating of the targetsite. A single energy source can exceed the thermal pain tolerance whenapplied from one side or location. When the energy is provided frommultiple sites, and/or when different sources of energy are used, thereis minimized risk to exceed the thermal pain tolerance. In oneembodiment, the thermal energy may be applied through multiple sources,as known in the art, e.g., radiofrequency, focused ultrasound, etc. Thethermal energy may be applied in a continuous manner, or it may beapplied in an oscillatory manner, or it may be applied in a pulsedmanner. Oscillatory or pulsed thermal energy application reduces thethermal damage to normal cells, while sufficiently heating cells towhich the nanoparticles are attached to the desired temperature. In thisembodiment, a preferred nanoparticle is supramagnetic nanoparticle orsupraparamagentic ferric oxide nanoparticle, nanotube, or nanowire.

In one embodiment, the thermal energy applied is an alternating magneticforce applied intermittently as an alternating magnet to heat to thenanoparticle-cell complex, and an imaging system measures thetemperature of the heated tissue and images the area involved. Thethermal imaging system (photoacoustic or MRI) controls the energydelivery from the unit to maintain the temperature at the target site at39° C.-48° C. The thermal imaging system (photoacoustic or MRI) controlsthe energy delivery from the unit to maintain the temperature at thetarget site at 37° C.-<60° C. The thermal imaging system (photoacousticor MRI) controls the energy delivery from the unit to maintain thetemperature at the target site at 37° C.-41° C. The thermal imagingsystem (photoacoustic or MRI) controls the energy delivery from the unitto maintain the temperature at the target site at 42° C.-46° C. Thethermal imaging system (photoacoustic or MRI) controls the energydelivery from the unit to maintain the temperature at the target site at47° C.-50° C. The thermal imaging system (photoacoustic or MRI) controlsthe energy delivery from the unit to maintain the temperature at thetarget site at 50° C.-58° C. If the nanoparticles are attached to tumorcells, the tumor cells are selectively or preferentially treated overnormal non-tumor cells. The thermal energy may be applied from multiplelocations covering at least 1°-90°, up to 180°, and greater than 180° ofthe circumference of the target tissue.

The method may be used to image and treat benign or malignant lesions,infective agents such as bacteria, fungi, parasites, viruses, prions,etc.

In one embodiment, the method may be used with preloaded stem cells,macrophages, dendritic cells or their exosomes to locate or track themthroughout the body, imaging their accumulation in vessels or tissues ata site of inflammation.

The nanoparticles may be synthetic, organic (e.g., liposomes),non-organic, non-magnetic, magnetic, paramagnetic, diamagnetic,supramagnetic, non-magnetic, mesoporous carbide-derived carbon, ironoxide nanoparticles with gold, graphene oxide and mesoporous siliconenanostructures, carbon, quantum dots, nanoshells, nanorods, nanotubes,nanowires, quantum dots, etc. Illustrative and non-limiting specificexamples also include liposomal nanoparticles, liposome-PEGnanoparticles, micellar polymeric platform nanoparticles, L-adeninenanoparticles, L-lysine nanoparticles, PEG-deaminase nanoparticles,polycyclodextrin nanoparticles, polyglutamate nanoparticles, calciumphosphate nanoparticles, antibody-enzyme conjugated nanoparticles,polymeric lipid hybrid nanoparticles, nanoparticles containing acombination of two-three elements such as gold, gold-iron oxide,iron-zinc oxide, metallic nanoparticles, polylacticglycolic acidnanoparticles, ceramic nanoparticles, silica nanoparticles, silicacrosslinked block polymer micelles, albumin-based nanoparticles,albumin-PEG nanoparticles, dendrimer attached magnetic or non-magneticnanoparticles, etc.

Any antibodies known in the art may be used, whether targeting normalcells or tissues or pathological cells or tissues, either at a fixedstatic site e.g., solid tumors, or not at a fixed static site, e.g.,circulating malignant cells such as blood cells, etc. Thus, thefollowing are exemplary and not limiting: abciximab, adalimuab,alemtuzumab, brentuximab, blimubab, canakinumab, alemtuzumab, cituximab,cetrolizumab, daclizumab, denosumab, efalizumab, gemtizumab, glimumab,lipilimumab, infliximab, rituximab, muromonab, oftamumab, palivizumab,panitumab, ranibizumab, tositumomab, rastuzumab, etc.

In one specific embodiment, iron oxide nanoparticles are coated witholigosaccharides for imaging and thermotherapy. In one specificembodiment, the nanoparticles are coated with thermosensitive polymerscarrying specific agents, e.g., anti-infectives, chemotherapeutics,anti-VEGFs, anti-EGFRs, hormones, immunosuppressants, immunostimulatoryagents, DNA, RNA, siRNA, genes, nucleotides, etc.

In one embodiment, nanoparticles are coated or otherwise associated withbiocompatible molecules. Exemplary and non-limiting biocompatiblemolecules include PEG, biotin, CPP, ACPP, dendrimers alone or conjugatedwith poly beta amine, small organic molecules, etc.

In one embodiment, enhanced cell penetration of theantibody-nanoparticle complex may be achieved usingpenetration-enhancing techniques. One example of such a penetrationenhancing technique is the application of ultrasound energy at thetarget site. One example of such a penetration enhancing technique isthe use of electroporation, as is known in the art, at the target site.A combination of ultrasound and electroporation may be used. Suchpenetration enhancing techniques advantageously reduce the total amountof energy applied to achieve the desired effect. The result is thatnormal cells are less affected or not affected by the therapy, whileproviding the desired amount, duration, extent, etc. of therapy to thetarget cells. The combination of CPP and poly beta amine is particularlybeneficial to enhance cell penetration.

In one embodiment, enhanced immunogenicity may be achieved using agentsthat control the cellular immune response to DNA damage. This embodimenthas utility particularly in patients undergoing cancer immunotherapy.Exemplary but not limiting agents that control the cellular immuneresponse to DNA damage include inhibitors to CHK1, CHK2, CHM1, and CHM2.In use, such agents are attached to the nanoparticles by methods knownin the art, e.g., using linkers, coatings, etc. At the target site, suchagents enhance the patient's immune response to malignant cells andfunction synergistically with the inventive method.

In one embodiment, cells are grown in culture with the nanoparticles.The nanoparticles are thus incorporated in the cells while in culture,and prior to injection into the body. They cells are thus associatedwith the nanoparticles and may be termed “nanoparticle preloaded”cultured cells. Exemplary and non-limiting cultured cell types includestem cells, macrophages, dendritic cells, lymphocytes, other leukocytes,dendritic cells, exosomes, etc. After injection of the nanoparticlepreloaded cells into the body, they may be traced and imaged in variousorgans. Exemplary and non-limiting organs include lung, heart, brain,the respiratory system, vessels, etc. In this embodiment inflammatoryprocess in tissues of an organ may be indirectly imaged.

In embodiment, the nanoparticles range from 1 nm-800 nm. In oneembodiment, the nanoparticles range from 20 nm to 200 nm. In oneembodiment, the nanoparticles range from 1 nm-20 nm. In one embodiment,the nanoparticles range from 1 nm-10 nm.

In one specific embodiment, the target site contains a lesion orpathology, and the nanoparticles contain a polymeric coating that itselfcontains a medication that is released when the temperature is 40°C.-47° C. In this specific embodiment, the lesion or pathology istreated by both thermally and by the medication.

In one specific embodiment, the patient is treated simultaneously orwith hemofiltration, hemoadsorption, mesoporous carbide-derived carbon,or another type of agent to prevent a cytokine storm.

In one embodiment, the method creates combinations of images to enhanceimage visibility, distinction, and utility. For example, the use ofphotoacoustic imaging with MRI, ultrasound, or light creates and moredistinct image of a structure without heating the structuresignificantly beyond 37° C.-39° C. Similarly, while an internalstructure cannot be imaged using radiofrequency wave energy or microwaveenergy alone, it is possible to create a photoacoustic image of aninternal structure with radiofrequency wave energy or microwave energyalone if an antibody-coated nanoparticle, with the antibody targetingthe nanoparticle to a tumor or structure such as endothelial cells,heart cells, muscle cells, or tumor cells, etc. are initially injected,e.g. are intravenously injected. Because nanoparticles of 1 nm aresignificantly smaller than a micron, two nanoparticles are separatedonly by <2 nm-3 nm, using thermal energy, they can create distinctseparate signals that can be electronically enhanced. Thus, usingphotoacoustic technology, one can calculate and measure two pointsseparated by that distance. This is beyond the optical resolution of astructure. The inventive method thus enhances the images obtained bylight, MRI, CT scan, PET scan etc.

Example

FIG. 2 illustrates a patient with a choroidal tumor, diagnosedclinically as malignant melanoma. The patient is treated with theinventive thermotherapy using ferromagnetic nanoparticles coated with(poly)ethylene glycol, anti-tumor antibody, and a polymer havinganti-cancer medication that releases the medication when the temperaturereaches 42° C. The ferromagnetic nanoparticles are also coated withradioactive molecules that emit α or β radiation with a short half-life(e.g., 3 hours to days). The nanoparticles are administeredintravenously to the patient. A natural magnet or an electromagnet issutured to the sclera if the tumor cannot be covered with the magnet ifplaced externally as shown in FIG. 2. If the tumor is locatedanteriorly, an electromagnet is preferably applied non-invasively,through the conjunctiva to the tumor as shown in FIG. 3. After a waitingperiod, e.g., one minute to one hour, sufficient additionalferromagnetic particles are accumulated in the tumor. After the magnetis removed, the patient is treated locally by the same electromagnetusing a rapidly alternating magnetic field. However, when it isdesirable to treat the tumor and possible metastatic lesions, a largereversible magnet covering the entire body is used and the patient'sentire body receives thermotherapy.

By rapidly reversing the magnetic field, the ferromagnetic nanoparticlesare heated due to an internal semicircular motion in the individualferromagnetic nanoparticles. The degree of heating is recorded by thephotoacoustic system which, in turn, is communicated by a processor withthe reversible magnet to generate the desired temperature at the tumorsite. When the desired temperature is achieved, this desired temperatureis maintained for a desired period, e.g., about 15 minutes in oneembodiment, greater than 15 minutes in another embodiment. In general,the initial thermal energy is delivered to achieve a temperature of 42°C. to 43° C. to release the drugs from the nanoparticles. If needed, thetemperature is gradually increased to, e.g., 45° C., 47° C., 50° C., orgreater than 50° C. for a period, as needed. Parameters such as theseare programmed by processor software that controls thermal energydelivery. FIG. 6 illustrates how information obtained from thephotoacoustic system controls the energy delivery by the processor toachieve and maintain the desired temperature, for a desired time, in thenanoparticle-cell complex at the tumor or lesion site.

After thermotherapy is completed, the patient is administered topicalantibiotics for a few days and remain under physician observation. Thetreatment may be repeated at intervals of 3 months to 9 months, or moreor less frequently as needed, to destroy the tumor. The release ofthermal energy, radioactivity, and drugs during the therapy generallygradually damages tumor cells and permits gradual patient recovery.Because the therapy is generally not invasive, or is only minimallyinvasive in embodiments where an internal magnet is used, the patientdoes not require long-term hospitalization. In a patient with severedisease, the patient may be treated with a low dose of anti-cancermedication to further kill the already damaged tumor cells, withoutdamaging healthy cells.

In one embodiment after placement of the magnet, the ferromagneticnanoparticles, which may additionally be radioactive and/or contain atherapeutic agent, are concentrated at a benign or malignant tumor aftersystemic or local administration at a localized site, e.g., in the eye,skin, breast, extremities, CNS, spinal cord, vertebra, pelvis,urogenital system, tongue, throat, etc. Examples are shown in FIGS. 4A,4B for a tumor affecting the lower leg and with a horseshoe-shapedelectromagnet configuration, and in FIG. 5 for a facial skin tumor.

In one embodiment, the treatment of the tumor requires medication only.In this embodiment, the ferromagnetic nanoparticles carry medication inthe absence of a radioactive molecule. The ferromagnetic nanoparticlesmay carry, e.g., immune modulators, anti-VEGF, sRNA, RNA, genes,bacteriophages, antibiotics, growth hormones, growth inhibitors, andcombinations of these as desired.

The references disclosed and cited below are expressly incorporated byreference herein in their entirety.

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Although several embodiments have been chosen to illustrate theinvention, those skilled in the art will readily appreciate that variouschanges and modifications can be made without departing from the scopeof the invention.

What is claimed is:
 1. A method of providing therapy, the methodcomprising administering to a patient in need thereof a plurality ofnanoparticles, the nanoparticles coated or otherwise associated with anantibody to specific cells, under conditions sufficient to permitantibody accumulation at a tissue target site, radiating the target sitewith an energy source to penetrate into the tissue target site tocontrollably heat the nanoparticles and generate thermal energy toinduce a photoacoustic signal or sound wave from the nanoparticles,using a processor to control the amount of thermal energy delivered atthe desired temperature to the target site, recording the temperatureand photoacoustic signal or sound wave from the target site or from oneor more multiple locations, and amplifying and processing the recordedphotoacoustic signal or sound waves to generate a computationaltomographic image of the nanoparticles at the tissue target site.
 2. Themethod of claim 1 where imaging generates high-resolution two- orthree-dimensional photoacoustic images.
 3. The method of claim 1 wherethe antibody is an anti-tumor antibody, the nanoparticles are attachedto tumor cells, and the method generates a two- or three-dimensionalphotoacoustic image of the tumor.
 4. The method of claim 1 where theantibody is directed to a receptor on a normal cell of an organ, thenanoparticles are attached to the normal cells, and the method generatesa two- or three-dimensional photoacoustic image of an organ regardlessof location of the organ in the body.
 5. The method of claim 1 whereimaging is by a modality selected from the group consisting ofradiofrequency, microwave, ultrasound, focused ultrasound, andcombinations thereof.
 6. The method of claim 5 where imaging is byphotoacoustic temperature imaging combined with a modality selected fromthe group consisting of ultrasound, focused ultrasound, magneticresonance imaging (MRI), functional MRI (fMRI), computed tomography(CT), positron emission tomography (PET), OCT, and alternating magneticfield imaging, resulting in enhanced image acquisition and enhancedresolution.
 7. The method of claim 1 where the processor communicatesthe temperature from the photoacoustic source to the energy source tocontrol the amount and duration of energy delivered to a desiredtemperature.
 8. The method of claim 1 where the photoacoustic signal orsound wave is recorded and produced as each of a thermal graph of thetarget site, and as an image in one-dimension, two-dimensions, orthree-dimensions.
 9. The method of claim 1 where the energy source toradiate the target site is selected from the group consisting ofelectromagnetic radiation, ultraviolet radiation, visible light,infrared light, radiofrequency waves, microwaves, focused ultrasound,alternating magnetic field radiation, and combinations thereof.
 10. Themethod of claim 1 where the energy is applied from multiple sites orusing multiple energy sources thus minimizing or preventing pain to thepatient and overheating of the target site.
 11. The method of claim 1where the energy is applied in a manner selected from the groupconsisting of continuous, intermittent, oscillatory, pulsed, andcombinations thereof.
 12. The method of claim 1 where the energy isapplied in an oscillatory or pulsed manner thus reducing thermal damageto normal cells while sufficiently heating cells to which thenanoparticles are attached to the desired temperature.
 13. The method ofclaim 1 where the energy is applied intermittently as an alternatingmagnetic force to heat to the nanoparticle-cell complex, and imagingmeasures the temperature of the heated tissue and images the targetsite.
 14. The method of claim 1 where the target site is maintained at atemperature selected from the group consisting of 39° C. to 48° C., 37°C. to <60° C., 37° C. to 41° C., 42° C. to 46° C., 47° C. to 50° C., and50° C. to 58° C.
 15. The method of claim 1 where the nanoparticles arecoated or otherwise associated with biocompatible molecules selectedfrom the group consisting of PEG, biotin, CPP, ACPP, dendrimers,dendrimers conjugated with poly beta amine, small organic molecules, andcombinations thereof.
 16. The method of claim 1 provided to the patientin need thereof with a method for enhanced cell penetration of theantibody-nanoparticle complex, the method selected from the groupconsisting of ultrasound, electroporation, and combinations thereof andresulting in therapy of cells at the target site while sparing normalcells.
 17. The method of claim 1 further comprising providing an agentto result in enhanced immunogenicity where the patient in need thereofis undergoing cancer immunotherapy, the agent selected from the groupconsisting of CHK1, CHK2, CHM1, and CHM2.
 18. The method of claim 1where the patient in need thereof is treated simultaneously or withhemofiltration, hemoadsorption, mesoporous carbide-derived carbon, oranother type of agent to prevent a cytokine storm.
 19. The method ofclaim 1 create a photoacoustic image of an internal structure withradiofrequency wave energy or microwave energy alone.
 20. The method ofclaim 10 performed using nanoparticles selected from the groupconsisting of supramagnetic ferric oxide nanoparticles,supraparamagentic ferric oxide nanoparticles, and combinations thereof.